Engineering Responses to Amino Acid Substitutions in the VP0- and VP3-Coding Regions of PanAsia-1 Strains of Foot-and-Mouth Disease Virus Serotype O.

The presence of sequence divergence through adaptive mutations in the major capsid protein VP1, and also in VP0 (VP4 and VP2) and VP3, of foot-and-mouth disease virus (FMDV) is relevant to a broad range of viral characteristics. To explore the potential role of isolate-specific residues in the VP0 and VP3 coding regions of PanAsia-1 strains in genetic and phenotypic properties of FMDV, a series of recombinant full-length genomic clones were constructed using Cathay topotype infectious cDNA as the original backbone. The deleterious and compensatory effects of individual amino acid substitutions at positions 4008 and 3060 and in several different domains of VP2 illustrated that the chain-based spatial interaction patterns of VP1, VP2, and VP3 (VP1-3), as well as between the internal VP4 and the three external capsid proteins of FMDV, might contribute to the assembly of eventually viable viruses. The Y2079H site-directed mutants dramatically induced a decrease in plaque size on BHK-21 cells and viral pathogenicity in suckling mice. Remarkably, the 2079H-encoding viruses displayed a moderate increase in acid sensitivity correlated with NH4Cl resistance compared to the Y2079-encoding viruses. Interestingly, none of all the 16 rescued viruses were able to infect heparan sulfate-expressing CHO-K1 cells. However, viral infection in BHK-21 cells was facilitated by utilizing non-integrin-dependent, heparin-sensitive receptor(s) and replacements of four uncharged amino acids at position 3174 in VP3 of FMDV had no apparent influence on heparin affinity. These results provide particular insights into the correlation of evolutionary biology with genetic diversity in adapting populations of FMDV.IMPORTANCE The sequence variation within the capsid proteins occurs frequently in the infection of susceptible tissue cultures, reflecting the high levels of genetic diversity of FMDV. A systematic study for the functional significance of isolate-specific residues in VP0 and VP3 of FMDV PanAsia-1 strains suggested that the interaction of amino acid side chains between the N terminus of VP4 and several potential domains of VP1-3 had cascading effects on the viability and developmental characteristics of progeny viruses. Y2079H in VP0 of the indicated FMDVs could affect plaque size and pathogenicity, as well as acid sensitivity correlated with NH4Cl resistance, whereas there was no inevitable correlation in viral plaque and acid-sensitive phenotypes. The high affinity of non-integrin-dependent FMDVs for heparin might be explained by the differences in structures of heparan sulfate proteoglycans on the surfaces of different cell lines. These results may contribute to our understanding of the distinct phenotypic properties of FMDV in vitro and in vivo.

Progress in the physiological and genetic mechanisms underlying the high prolificacy of the Erhualian pig.

The Erhualian pig, originally distributed in the Taihu area, is well known for its universally high fertility. Previous studies have found that high ovulation numbers, low embryo mortality and high uterine volumes are important physiological characteristics underlying the high prolificacy of the Erhualian pig. Although candidate genes such as follicle-stimulating hormone ß (FSHß) and several quantitative trait loci (QTLs) on chromosome 2, 6, 7, 8, 12, 13 and 15 have been reported to affect the litter size in the Erhualian pig, the key genes related to high prolificacy remain poorly understood. In this review, we summarize the recent research progress in the physiological and genetic mechanisms underlying the high prolificacy of the Erhualian pig. First we review the role of high ovulation numbers, low embryo mortality rates and high uterine volumes in the formation of the high litter size in the Erhualian pig. Then we summarize candidate genes and QTLs for the high litter size detected by classical strategies, as well as by genomic strategies. Moreover, we describe the methods to investigate the causative genes of the high prolificacy through integrative analysis of multi-omics data including genomics, transcriptomics, proteomics and functional genomics. This review will provide insights to understand the molecular basis of the high prolificacy in the Erhualian pig.

Comparative transcriptome analysis of developmental stages of the Limonium bicolor leaf generates insights into salt gland differentiation.

With the expansion of saline land worldwide, it is essential to establish a model halophyte to study the salt-tolerance mechanism. The salt glands in the epidermis of Limonium bicolor (a recretohalophyte) play a pivotal role in salt tolerance by secreting excess salts from tissues. Despite the importance of salt secretion, nothing is known about the molecular mechanisms of salt gland development. In this study, we applied RNA sequencing to profile early leaf development using five distinct developmental stages, which were quantified by successive collections of the first true leaves of L. bicolor with precise spatial and temporal resolution. Specific gene expression patterns were identified for each developmental stage. In particular, we found that genes controlling salt gland differentiation in L. bicolor may evolve in a trichome formation, which was also confirmed by mutants with increased salt gland densities. Genes involved in the special ultrastructure of salt glands were also elucidated. Twenty-six genes were proposed to participate in salt gland differentiation. Our dataset sheds light on the molecular processes underpinning salt gland development and thus represents a first step towards the bioengineering of active salt-secretion capacity in crops.

Isolation, Culture and Identification of Porcine Skeletal Muscle Satellite Cells.

The objective of this study was to establish the optimum protocol for the isolation and culture of porcine muscle satellite cells. Mononuclear muscle satellite cells are a kind of adult stem cell, which is located between the basal lamina and sarcolemma of muscle fibers and is the primary source of myogenic precursor cells in postnatal muscle. Muscle satellite cells are a useful model to investigate the mechanisms of muscle growth and development. Although the isolation and culture protocols of muscle satellite cells in some species (e.g. mouse) have been established successfully, the culture system for porcine muscle satellite cells is very limited. In this study, we optimized the isolation procedure of porcine muscle satellite cells and elaborated the isolation and culture process in detail. Furthermore, we characterized the porcine muscle satellite cells using the immunofluorecence. Our study provides a reference for the isolation of porcine muscle satellite cells and will be useful for studying the molecular mechanisms in these cells.

Resiquimod and polyinosinic-polycytidylic acid formulation with aluminum hydroxide as an adjuvant for foot-and-mouth disease vaccine.

BACKGROUND: Toll-like receptor (TLR) agonists reportedly have potent antiviral and antitumor activities and may be a new kind of adjuvant for enhancing immune efficacy. Resiquimod (R848) is an imidazoquinoline compound with potent antiviral activity and functions through the TLR7/TLR8 MyD88-dependent signaling pathway. Polyinosinic-polycytidylic acid [poly(I:C)] is a synthetic analog of double-stranded RNA that induces the production of pro-inflammatory cytokines by the activation of NF-κB through TLR3. This study investigated the potential of R848 and poly(I:C) as an adjuvant 146S foot-and-mouth disease virus (FMDV) vaccine formulated with aluminum hydroxide (Al(OH)3). RESULTS: Antibody titers to FMDV and CD8+ T cells were markedly enhanced in mice immunized to 146S FMDV + Al(OH)3 + R848 + poly(I:C) compared with mice immunized to FMDV + ISA206. IFN-γ secretion substantially increased compared with IL-4 secretion by splenic T cells stimulated with FMDV antigens in vitro, suggesting that R848, poly(I:C), and with Al(OH)3 together biased the immune response toward a Th1-type direction. CONCLUSIONS: These results indicated that the R848 and poly(I:C) together with Al(OH)3 enhanced humoral and cellular immune responses to immunization with 146S FMDV antigens. Thus, this new vaccine formulation can be used for FMDV prevention.

[Prevalence and causes of blindness and moderate and severe visual impairment among adults aged 50 years or above in Yongchuan District of Chongqing City: the China Nine-Province survey].

OBJECTIVE: To investigate the prevalence and causes of blindness and moderate and severe visual impairment among adults aged 50 years or above in Yongchuan of Chongqing City, China. METHODS: It was a population-based cross-section study.Geographically defined cluster sampling was used in randomly selecting 5663 individuals aged ≥ 50 years in Yongchuan District. The survey was preceded by a pilot study where operational methods were refined and quality assurance evaluation was carried out. All participants were enumerated through village registers followed door-to-door visits.Eligible individuals were invited to receive visual acuity measurement and eye examination. Prevalence of blindness and moderate and severe visual impairment was calculated according to different age, gender or education. And the reasons of blindness were analyzed.Statistical analyses were performed using Stata/SE Statistical Software, release 9.0. Chi-square test was used to investigate the association of age, gender and education with presenting and best corrected visual acuity. RESULTS: Five thousands six hundreds and sixty-three individuals were enumerated and 5390 persons were examined, the response rate was 95.18%. Based on the criteria of World Health Organization visual impairment classification in 1973, the prevalence of blindness and moderate and severe visual impairment defined as best corrected visual acuity was 2.12% (114/5390) and 5.40% (291/5390) respectively. The prevalence of blindness and moderate and severe visual impairment defined as presenting visual acuity was 2.49% (134/5390) and 10.71% (577/5390) respectively. The prevalence of blindness and moderate and severe visual impairment was higher in aged (trend χ(2) = 951.32, P = 0.000) , female (χ(2) = 33.35, P = 0.000) and illiterate (trend χ(2) equals; 141.32, P = 0.000) persons. Cataract was still the first leading cause of blindness and visual impairment.Un-corrected refractive error also was the main cause of visual impairment. CONCLUSIONS: The prevalence of blindness and moderate and severe visual impairment is relatively higher among older adults aged 50 years or above in Yongchuan District. The first leading cause of blindness and visual impairment is still cataract.

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.

[Expression of glial fibrillary acidic protein in retina of rats in acute experimental autoimmune encephalomyelitis].

OBJECTIVE: To investigate the effects of glial fibrillary acidic protein on blood-retinal barrier of rats in acute experimental autoimmune encephalomyelitis (EAE). METHODS: On the 8 day the 14 day and the 21 day after rats EAE model were developed, the function of BRB was observed by injected with Evans blue intravenously and the expression of GFAP was detected by immunohistochemistry. RESULTS: At various time points, the amount of BRB leaked into retina was obvious lower in the control group than those in EAE group (P < 0.01); GFAP immunoreactivity was limited to ganglion cell layer and nerve fiber layer in the control retina; In EAE group, GFAP immunoreactivity was significantly increased in ganglion cell layer and nerve fiber layer, and a little GFAP immunoreactivity was found in inner plexiform layer on post-immune (p.i.) day 8; On p.i. day 14, GFAP immunoreactivity was espressed either in ganglion cell layer and nerve fiber layer or in inner plexiform layer and inner nuclear layer, moreover transfixed in inner nuclear layer and outer nuclear layer; On p.i. day 21, GFAP immunoreactivity was limited to ganglion cell layer, nerve fiber layer, inner plexiform layer and inner nuclear layer. CONCLUSION: Acute EAE cause the rat's BRB damage that is possibly related to the activation of astrocyte.

Investigation of the efficiency of intrastromal ring segments with cross-linking using different sequence and timing for keratoconus.

AIM: To evaluate and compare the efficacy and stability of intrastromal corneal ring segment (ICRs) implantation with cross-linking (CXL) using different sequence and timing. METHODS: In this single retrospective study, 86 keratoconic eyes subjected the ICRs implantation. We analyzed only 41 eyes that had complete follow-ups. They were divided into three groups: ICRs implantation was applied only (group normal), ICRs first followed by CXL immediately (group CXL-S), CXL first followed by ICRs long after (group CXL-B). The visual acuity, refractive results, keratometry were compared preoperatively and 1y postoperatively. Their differences among the three groups were also analyzed. RESULTS: Group normal comprised 25 eyes, group CXL-S 8 eyes, and group CXL-B 8 eyes. There were improvements in the mean uncorrected distance visual acuity (UDVA) and the mean corrected distance visual acuity (CDVA) compared preoperatively and 1y postoperatively {UDVA: 0.31 (P=0.030) logarithmic minimum angle of resolution [logMAR] group normal, 0.4 (P=0.020) group CXL-S, 0.45 (P=0.001) group CXL-B; CDVA: 0.21 logMAR (P=0.013) group normal, 0.30 (P=0.036) group CXL-S; 0.26 (P=0.000) group CXL-B}. The refractive and topographic outcomes also showed improvements. In terms of comparisons among the three groups, all the P values were above 0.05, showing no significant difference. But only group CXL-B had improvement in UDVA and CDVA for all the patients. CONCLUSION: With safety and good visual outcomes, ICRs implantation is a viable alternative for keratoconus. No significant difference was found among these three groups.

The association between matrix metalloprotease-9 gene polymorphisms and primary angle-closure glaucoma in a Chinese Han population.

AIM: To examine the association between the single nucleotide polymorphisms (SNPs) of matrix metalloprotease-9 (MMP-9) gene and primary angle-closure glaucoma (PACG) in a Chinese Han population. METHODS: DNA samples were extracted from peripheral-blood mononuclear cells of 214 PACG patients and 224 healthy controls. Genotyping of rs3918249, rs3918254, rs17577 and rs3787268 in MMP-9 was performed using polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) analysis and the direct sequencing technique. The association between these genetic polymorphisms and risk of PACG was estimated by χ (2) test. RESULTS: The distributions of rs3918249, rs3918254, rs17577 and rs3787268 genotypes among cases and healthy controls were compatible with that from Hardy-Weinberg equilibrium (HWE, P>0.05).The increased frequency of CC and CT genotypes of rs3918254 were observed in PACG patients compared to healthy controls [P=0.006, Pcorrected (Pcorr)=0.048]. The haplotype analysis showed that the CCGG haplotype was nominal associated with PACG (P=0.015), however, the significant was lost when the Bonferroni correction was used (Pcorr=0.105). CONCLUSION: Our results revealed that rs3918254 in MMP-9 may be a susceptible locus to PACG in China, people with the CC and CT genotypes of rs3918254 are more susceptible to PACG. The susceptibility to PACG in Chinese Han patients may be not influenced by SNPs rs3918249, rs3787268 and rs17577 in MMP-9.

Study on Content Determination and Pharmacokinetics of Paeoniflorin and Paeonolide from Paeoniae Radix Alba in Rat Plasma / 中国中医药信息杂志

Objective To establish a method for the content determination of paeoniflorin and paeonolide in rat plasma by HPLC-MS/MS; To study its characteristics of pharmacokinetics.Methods HPLC-MS/MS method was adopted. The column was Ultimate RXB-C18 (2.1 mm×100 mm, 3 μm) with mobile phase consisted of acetonitrile- 0.1％ formic acid water solution. Flow rate was 0.3 mL/min; column temperature was 30℃; the volume was 5 μL; detection wavelength was 230 nm. Quantitative ions were m/z 525→120.9 (paeoniflorin and paeonolide), m/z 433→224.8 (geniposide). 8 SD male rats were collected blood 0.5 mL from orbital vein 0, 5, 10, 20, 30, 45, 60, 90, 120, 180, 360, 540, 720 min after intragastric administrationBaishaoDecoction 6 g (medicinal materials)/kg to determine the blood concentration of medicine. DAS V2.1.1 software was employed to calculate pharmacokinetic parameters. Results Good linearity was found in the range of 40–8000 ng/mL and 40–10000 ng/mL for paeoniflorin and paeonolide (r=0.9996,r=0.9995). The intra-day and inter-day RSD were less than 6％. The extraction recoveries were 92.33％–95.43％ and 88.65％–95.62％. The matrix effect values were 99.29％–107.86％ and 93.79％–104.05％.All items of method validation were in line with the requirements. The main pharmacokinetic parameters of paeoniflorin and paeonolide were showed as follows: Cmax of (8.50±2.01) and (4.13±0.72) μg/mL; tmax of (10.00± 1.73) and (20.00±2.11) min; t1/2 of (142.98±30.11) and (127.68±35.74) min.Conclusion The HPLC-MS/MS method established in this study is simple, feasible, accurate, reliable, highly sensitive, and specific, which can be applied to study the pharmacokinetic characteristics of paeoniflorin and paeonolide in rats in vivo.

[Connective tissue growth factor mediates high glucose-induced down-regulation of podocalyxin expression in mouse podocytes].

OBJECTIVE: To detect the effect of connective tissue growth factor (CTGF) on podocalyxin expression in mouse podocytes exposed to high glucose in vitro and explore the possible pathway involved. METHODS: The expression vector carrying a small interfering RNA (siRNA) targeting CTGF was transfected into mouse podocytes cultured in the presence of 1 g/L glucose (normal control), 4.5 g/L glucose (high glucose group), 1 g/L glucose + 3.5 g/L mannitol (iso-osmolar control group). The changes in the protein expression levels of podocalyxin, CTGF and ERK1/2 in the cells in response to the treatments were investigated using Western blotting. RESULTS: High glucose exposure for 24 and 48 h resulted in significantly decreased expression of podocalyxin and increased CTGF in the podocytes (P<0.05). Phosphorylation of ERK1/2 occurred as early as 30 min after the exposure, and the activation was maintained till 24 h. Transfection of the cells with siRNA targeting CTGF significantly inhibited these changes. CONCLUSION: CTGF is an important mediator of high glucose-induced podocyte damage and decreases the protein level of podocalyxin by the ERK1/2 pathway. CTGF-specific siRNA can alleviate high glucose-induced podocyte injury, suggesting its potential value in treatment of diabetic nephropathy.

The efficacy of FMD vaccine reduced non-structural proteins with a mAb against 3B protein.

A monoclonal antibody, 3BIgG, against the prokaryotically expressed foot-and-mouth disease virus (FMDV) non-structural protein (NSP) 3B was obtained. The 3BIgG-sepharose conjugant (3BmAb-6BFF) was prepared by adding the purified 3BIgG into epoxy-activated sepharose 6BFF, incubating with the inactivated FMDV, and then removing the sepharose by centrifugation. The vaccine was made from the supernatant emulsified with oil-adjuvant ISA206. Ten guinea pigs, 26 pigs and six cattle were vaccinated, and a vaccination control group was included without treatment with 3BmAb-6BFF. After 28 days, 9/10 pigs challenged with FMDV were protected, this result was the same as the control group, indicating that the vaccine potency was not reduced after treatment with 3BmAb-6BFF. The other animals were vaccinated weekly for nine weeks, and serum samples were collected to detect 3ABC-antibody titers. The results showed that 3ABC-antibody production was delayed and the positive antibody rates were lower when vaccination was carried out using vaccines treated with 3BmAb-6BFF compared with untreated vaccines. The findings of this study suggest that it is possible to reduce NSPs using a mAb-sepharose conjugant in FMD vaccines without reducing their efficacy.