Modification of the second translation initiation site restricts the replication of foot-and-mouth disease virus in PK-15 cells.

The translation initiation of foot-and-mouth disease virus (FMDV) occurs at two alternative initiation sites (Lab AUG and Lb AUG). Usually, the Lb AUG is more favorably used to initiate protein synthesis than the Lab AUG. To explore the effect of Lb AUG on FMDV replication and obtain FMDV with restricted replication, this initiation codon was mutated to a variety of non-AUG codons (UGG, AUC, CUG, and AAA). Fortunately, the modifications did not prevent viral viability but influenced replication characteristics of some FMDV mutants in a cell-specific manner, as was shown by the similar replication in BHK-21 cells and delayed growth kinetics in PK-15 cells. This attenuated phenotype of FMDV mutants in PK-15 cells was found to be correlated with reduced abilities to cleave eIF4GI and suppress interference (IFN) expression. As leader (L) protein was reported to be responsible for eIF4GI cleavage and inhibition of IFN expression, the in vivo L protein synthesis was examined during the infection of FMDV mutants. Our results showed that not only the total yield of L proteins was severely influenced but also the individual yield of L protein was seen to be affected, which implied that both the relative usage of the two initiation sites and overall translation efficiency were changed by Lb AUG modifications. In addition, the in vitro translation activity was also negatively regulated by Lb AUG mutations. Collectively, these findings suggested that the restricted replications of Lb AUG-modified FMDVs were related to the delayed eIF4GI cleavage and decreased ability to block IFN expression but were mainly determined by the inefficient translation initiation. FMDVs precisely with modifications of Lb AUG initiation codon may represent safer seed viruses for vaccine production. KEY POINTS: • The polyprotein translation of FMDV initiates at two alternative initiation sites (Lab AUG and Lb AUG). In order to explore the effect of Lb AUG on FMDV replication and obtain FMDV with restricted replication, the Lb initiation AUG was mutated to a variety of non-AUG codons (UGG, AUC, CUG, and AAA), and four FMDV mutants with Lb AUG modification were generated. • We found that partial FMDV mutants grew almost as well as WT virus in BHK-21 cells, a typical cell line used for FMD vaccine production, but displayed impaired replication in IFN-competent PK-15 cells. • The attenuation of mutant FMDVs in PK-15 cells was found to be correlated with delayed eIF4GI cleavage and decreased ability to block IFN expression. • We proved that the attenuated phenotype of Lb AUG-modified FMDVs was mainly determined by the inefficient translation initiation, as demonstrated by the decrease of total yield of L proteins and individual production of L protein. • We successfully generated genetically engineered FMDV with attenuated phenotype. The approach of precise engineering of FMDV with the modification of initiation codon provides a safe platform to produce inactivated antigen vaccines.

Comparison of Ultrasound-Guided Percutaneous Polidocanol Injection Versus Percutaneous Ethanol Injection for Treatment of Benign Cystic Thyroid Nodules.

OBJECTIVES: We compared the efficacy, safety, and cost-effectiveness of ultrasound-guided percutaneous polidocanol injection and percutaneous ethanol injection for the treatment of benign cystic and predominantly cystic thyroid nodules. METHODS: A total of 135 cystic thyroid nodules treated by percutaneous ethanol injection and 136 cystic thyroid nodules treated by percutaneous polidocanol injection were enrolled retrospectively in this study from May 2010 to March 2016. The nodules were followed after 1, 3, 6, and 12 months. Nodule volumes, symptoms scores, and cosmetic scores were assessed before treatment and at follow-up. The therapeutic success rate, safety, and cost-effectiveness between the groups were also compared. RESULTS: No significant differences in the reduction of the nodule volume, volume reduction rate, and therapeutic success were observed between the groups with cystic and predominantly cystic thyroid nodules during follow-up (P > .05). Neither the cosmetic scores (P = .59; P = .42) nor the symptom scores (P = .32; P = .73) in the cystic and predominantly cystic nodules were significantly different between the groups at the last follow-up. The complication rates for ethanol were higher than those for polidocanol (P < .05). However, the cost of polidocanol injection was higher than that of ethanol injection for cystic thyroid nodules (mean ± SD, US$97.18 ± US$22.17 versus US$43.36 ± US$5.51; P < .01). CONCLUSIONS: Ultrasound-guided percutaneous polidocanol injection can be an alternative for sclerotherapy of cystic or predominantly cystic thyroid nodules. However, its cost was higher than that of percutaneous ethanol injection.

Single Amino Acid Substitutions Surrounding the Icosahedral Fivefold Symmetry Axis Are Critical for Alternative Receptor Usage of Foot-and-Mouth Disease Virus.

The integrins function as the primary receptor molecules for the pathogenic infection of foot-and-mouth disease virus (FMDV) in vivo, while the acquisition of a high affinity for heparan sulfate (HS) of some FMDV variants could be privileged to facilitate viral infection and expanded cell tropism in vitro. Here, we noted that a BHK-adapted Cathay topotype derivative (O/HN/CHA/93tc) but not its genetically engineered virus (rHN), was able to infect HS-positive CHO-K1 cells and mutant pgsD-677 cells. There were one or three residue changes in the capsid proteins of O/HN/CHA/93tc and rHN, as compared with that of their tissue-originated isolate (O/HN/CHA/93wt). The phenotypic properties of a set of site-directed mutants of rHN revealed that E83K of VP1 surrounding the fivefold symmetry axis was necessary for the integrin-independent infection of O/HN/CHA/93tc. L80 in VP2 was essential for the occurrence of E83K in VP1 during the adaptation of O/HN/CHA/93wt to BHK-21 cells. L80M in VP2 and D138G in VP1 of rHN was deleterious, which could be compensated by K83R of VP1 for restoring an efficient infection of integrin-negative CHO cell lines. These might have important implications for understanding the molecular and evolutionary mechanisms of the recognition and binding of FMDV with alternative cellular receptors.

Efficacy and safety of ultrasound-guided percutaneous polidocanol sclerotherapy in benign predominantly cystic thyroid nodules: a prospective study.

OBJECTIVE: To evaluate the efficacy and safety of percutaneous polidocanol injection (PPI) in treatment of predominantly cystic thyroid nodules. MATERIALS AND METHODS: This prospective study included 111 patients with 122 benign predominantly cystic thyroid nodules inducing pressure symptoms or cosmetic problems. The nodules were randomized to a single aspiration with (n = 61) or without (n = 61) subsequent PPI and followed up after 1, 3, 6, and 12 months. Ten patients (12 nodules) declined to follow up after aspiration in group 2. Nodule volumes, symptoms scores, and cosmetic scores were evaluated before and after treatment. The therapeutic success rate and safety of PPI for treatment of predominantly cystic thyroid nodules were also evaluated. RESULTS: In the PPI group, the nodule volumes were reduced from 13.67 ± 9.90 to 2.60 ± 2.66 (p < .001). Therapeutic success rate (nodule volume reduction >50%) was obtained in 57 of 61 (93.44%) nodules in the PPI group, compared to seven of 49 (14.29%) in the aspiration group (p < .001). In the aspiration group, the nodule volume was not significantly reduced. The reduction in symptom scores was significantly higher in the PPI group (from 3.60 ± 1.65 to 1.60 ± 1.19) than in the aspiration group (from 3.62 ± 1.89 to 3.30 ± 1.06) (p < .001, between groups). The reduction in cosmetic scores showed a significant difference between groups (p < .001). In total, 4.92% of patients (3/61) in the PPI group and 85.71% (42/49) in the aspiration group showed recurrence during the follow-up period. There was a significant difference in the recurrence rate between groups (p < .001). No major side-effects occurred. CONCLUSIONS: US-guided PPI of benign recurrent predominantly cystic thyroid nodules is effective and safe. PPI is an important alternative to benign recurrent predominantly cystic thyroid nodules.

Growth of hollow cell spheroids in microbead templated chambers.

Cells form hollow, spheroidal structures during the development of many tissues, including the ocular lens, inner ear, and many glands. Therefore, techniques for in vitro formation of hollow spheroids are valued for studying developmental and disease processes. Current in vitro methods require cells to self-organize into hollow morphologies; we explored an alternative strategy based on cell growth in predefined, spherical scaffolds. Our method uses sacrificial, gelatin microbeads to simultaneously template spherical chambers within a hydrogel and deliver cells into the chambers. We use mouse lens epithelial cells to demonstrate that cells can populate the internal surfaces of the chambers within a week to create numerous hollow spheroids. The platform supports manipulation of matrix mechanics, curvature, and biochemical composition to mimic in vivo microenvironments. It also provides a starting point for engineering organoids of tissues that develop from hollow spheroids.

Sox10+ adult stem cells contribute to biomaterial encapsulation and microvascularization.

Implanted biomaterials and biomedical devices generally induce foreign body reaction and end up with encapsulation by a dense avascular fibrous layer enriched in extracellular matrix. Fibroblasts/myofibroblasts are thought to be the major cell type involved in encapsulation, but it is unclear whether and how stem cells contribute to this process. Here we show, for the first time, that Sox10+ adult stem cells contribute to both encapsulation and microvessel formation. Sox10+ adult stem cells were found sparsely in the stroma of subcutaneous loose connective tissues. Upon subcutaneous biomaterial implantation, Sox10+ stem cells were activated and recruited to the biomaterial scaffold, and differentiated into fibroblasts and then myofibroblasts. This differentiation process from Sox10+ stem cells to myofibroblasts could be recapitulated in vitro. On the other hand, Sox10+ stem cells could differentiate into perivascular cells to stabilize newly formed microvessels. Sox10+ stem cells and endothelial cells in three-dimensional co-culture self-assembled into microvessels, and platelet-derived growth factor had chemotactic effect on Sox10+ stem cells. Transplanted Sox10+ stem cells differentiated into smooth muscle cells to stabilize functional microvessels. These findings demonstrate the critical role of adult stem cells in tissue remodeling and unravel the complexity of stem cell fate determination.