Improvement of enzymatic stability and intestinal permeability of deuterohemin-peptide conjugates by specific multi-site N-methylation.

The deuterohemin-peptide conjugate, DhHP-6 (Dh-ß-AHTVEK-NH(2)), is a microperoxidase mimetic, which has demonstrated substantial benefits in vivo as a scavenger of reactive oxygen species (ROS). In this study, specific multi-site N-methylated derivatives of DhHP-6 were designed and synthesized to improve metabolic stability and intestinal absorption, which are important factors for oral delivery of therapeutic peptides and proteins. The DhHP-6 derivatives were tested for (1) scavenging potential of hydrogen peroxide (H(2)O(2)); (2) permeability across Caco-2 cell monolayers and everted gut sacs; and (3) enzymatic stability in serum and intestinal homogenate. The results indicated that the activities of the DhHP-6 derivatives were not influenced by N-methylation, and that tri-N-methylation of DhHP-6 could significantly increase intestinal flux, resulting in a two- to threefold higher apparent permeability coefficient. In addition, molecules with N-methylation at selected sites (e.g., Glu residue) showed high resistance against proteolytic degradation in both diluted serum and intestinal preparation, with 50- to 140-fold higher half-life values. These findings suggest that the DhHP-6 derivatives with appropriate N-methylation could retain activity levels equivalent to that of the parent peptide, while showing enhanced intestinal permeability and stability against enzymatic degradation. The tri-N-methylated peptide Dh-ß-AH(Me)T(Me)V(Me)EK-NH(2) derived from this study may be developed as a promising candidate for oral administration.

N isotopes and N cycle in the TieShanPing subtropical forest ecosystem, Southwestern China.

Nitrogen is essential for forest growth and forest stand development. It is commonly a limited factor for forest productivity. We examined delta (15)N values in soils and plants by studying the sources of N used by vegetation and cycles of N in a 43-year-old plantation of the TieShanPing forest ecosystem in southwestern China, dominated by massone pine (Pinus massoniana). The N concentration of plant materials ranges from 1.1% to 2.2%. The nitrogen concentration of P. massoniana was 1.3% while soils showed the concentration of 0.04-0.15%. Regarding natural abundance of (15)N, large significant variation (-6.0 per thousand to -3.8 per thousand) in delta (15)N values was observed among shrub and tree leaves. delta (15)N values were also significantly varied from -4.7 per thousand to -3.8 per thousand among the pioneer species in the plantation. Soil delta (15)N values (3.1-6.3 per thousand) were significantly enriched compared to those values in plant samples. Despite the negative delta (15)N values of the vegetation cover, the high delta (15)N values in the topsoil indicate that return of N to soils by litter-fall is minimal on TieShanPing and the present forests do not change very much the soil (15)N signals at the surface layer. The positive delta (15)N values may also indicate large N losses from the soil system vial leaching, volatilization and plant uptake.

Identification of a missense mutation causing exon skipping in a neurofibromatosis type 1 patient.

Neurofibromatosis type 1 (NF1), caused by germ line mutations of the NF1 tumor-suppressor gene, is one of the most common autosomal dominant disorders. Here, we reported a NF1 patient with the mutation NF1 c.4367+1G>C. This sequence change locates at the first nucleotide of NF1 intron 32 within the consensus splice site. Compared with NF1 c.4367G>C predicted to potentially damage the wild-type donor site at c.4367, the NF1 c.4367+1G>C potentially abolishes this wild-type donor site by in silico analysis. In vitro minigene assay revealed that the NF1 c.4367+1G>C may cause exon 32 skipping. Our result provides further evidence for its clinical significance of NF1 c.4367+1G>C in clinical practise.

[Emissions Inventory and Characteristics of NOx from Cement Industry].

Activity data and technical information of cement production lines in China from 2011 to 2015 were collected. A NOx emissions calculating model and emissions database were developed for the cement industry, and the NOx emissions characteristics of cement production lines in China from 2011 to 2015 were analyzed. The results showed that NOx emissions increased from 1.69 million tons in 2010 to 1.99 million tons in 2012, and then decreased in the subsequent three years to 1.68 million tons in 2015. The provincial-level emissions are significantly uneven. Anhui, Sichuan, Henan, Hunan, Yunnan, and Shandong provinces rank among the top six contributors in the country and together accounted for about 40% of the national emissions in 2015. Shanghai, Inner Mongolia, Shanxi, Xinjiang, Hunan, Yunnan, and Sichuan were the top seven by emissions factor. Lines with capacity of more than 4000 t·d-1 contributed the largest part of NOx emissions. The average NOx emissions factor of more than 4000 t·d-1 lines was 1.24 kg·t-1. The rapid spread of pre-calcining kilns in China and a higher pollution abatement level were the main factors leading to variations in NOx pollution characteristics in China.

Antiviral activity of a zymolytic grain based extract on human immunodeficiency virus type 1 in vitro.

Increasing evidence shows that grains may play a role in disease prevention beyond the simple provision of energy and nutrients. It has been reported that some components contained in grains exert their functional effects on viral and bacterial infections and protect against various cancers. However, until now, hardly any intervention studies have investigated the effects of grains or grain based extracts on the inhibition of HIV-1 infection. In this study, the antiviral function of a zymolytic grain based extract (ZGE) was detected in vitro and in rats, and the antiviral mechanism was investigated. Results showed that ZGE had an inhibition effect on HIV-1 infection in vitro with low cytotoxic effects. The study of the mechanism demonstrated that this functional food possibly acted on the viral surface structure protein gp120 which is responsible for cell binding, as well as on the postattachment stage of the virus. The sera of model rats administrated with this food by gavage presented anti-infection abilities against HIV-1 in vitro during a serum concentration associated period of time. These findings provide valuable insights into the application of ZGE on the control of viral load, which may contribute to future anti-HIV treatment with less adverse effects.

Detection of human papillomavirus genotypes associated with mucopurulent cervicitis and cervical cancer in Changchun, China.

OBJECTIVE: To determine the prevalence and genotype distribution of human papillomavirus (HPV) infection among Chinese patients with mucopurulent cervicitis (MPC) or cervical cancer (CC). METHODS: In total, 191 cases of CC (n=66), acute MPC (n=84), and healthy cervix controls (n=41) were initially included; samples were collected between May 21, 2008, and October 9, 2011. Cervical specimens were screened for HPV using a nested polymerase chain reaction assay and DNA sequencing. RESULTS: Overall prevalence of HPV infection was 20.0% in the control group, 53.3% in the MPC group, and 93.8% in the CC group. The predominant genotype detected in all 3 groups was the oncogenic variant HPV 16 (87.7%, 18.7%, and 10.0% in the CC, MPC and control specimens, respectively). The second most frequent genotype among patients with MPC was HPV 58. This variant is also oncogenic and was detected at a higher rate in the MPC group (9.3%) than in the control (2.5%) and CC (1.5%) groups. CONCLUSION: Infection with HPV was prevalent among Chinese women with MPC or CC. Furthermore, the high prevalence of oncogenic genotypes observed among HPV-positive patients with MPC suggests that this group is at increased risk of developing CC.

Single domain antibody multimers confer protection against rabies infection.

Post-exposure prophylactic (PEP) neutralizing antibodies against Rabies are the most effective way to prevent infection-related fatality. The outer envelope glycoprotein of the Rabies virus (RABV) is the most significant surface antigen for generating virus-neutralizing antibodies. The small size and uncompromised functional specificity of single domain antibodies (sdAbs) can be exploited in the fields of experimental therapeutic applications for infectious diseases through formatting flexibilities to increase their avidity towards target antigens. In this study, we used phage display technique to select and identify sdAbs that were specific for the RABV glycoprotein from a naïve llama-derived antibody library. To increase their neutralizing potencies, the sdAbs were fused with a coiled-coil peptide derived from the human cartilage oligomeric matrix protein (COMP48) to form homogenous pentavalent multimers, known as combodies. Compared to monovalent sdAbs, the combodies, namely 26424 and 26434, exhibited high avidity and were able to neutralize 85-fold higher input of RABV (CVS-11 strain) pseudotypes in vitro, as a result of multimerization, while retaining their specificities for target antigen. 26424 and 26434 were capable of neutralizing CVS-11 pseudotypes in vitro by 90-95% as compared to human rabies immunoglobulin (HRIG), currently used for PEP in Rabies. The multimeric sdAbs were also demonstrated to be partially protective for mice that were infected with lethal doses of rabies virus in vivo. The results demonstrate that the combodies could be valuable tools in understanding viral mechanisms, diagnosis and possible anti-viral candidate for RABV infection.

A novel disulfide-stabilized single-chain variable antibody fragment against rabies virus G protein with enhanced in vivo neutralizing potency.

Rabies is a fatal infectious disease requiring efficient protection provided by post-exposure prophylaxis (PEP) with rabies immunoglobulin (RIG). The single-chain Fv fragment (scFv) is a small engineered antigen binding protein derived from antibody variable heavy (V(H)) and light (V(L)) chains. This novel antibody format may potentially replace the current application of RIG to detect and neutralize rabies virus (RV). However, the broad use of scFvs is confined by their generally low stability. In this study, a scFv (FV57) was constructed based on the monoclonal antibody, MAB57, against RV. To enhance its stability and neutralizing potency, a disulfide-stabilized scFv, ds-FV57, was also derived by introduction of cysteines at V(H)44 and V(L)100. Furthermore, the cysteine at V(L)85 of ds-FV57 was mutated to serine to construct ds-FV57(VL85Ser) in order to avoid potential mis-formed disulfide bonds which would alter the affinity of the scFv. The stability and activity of all three proteins expressed in Escherichia coli were evaluated. All of the constructed scFvs could provide efficient protection against RV infection both in vivo and in vitro. However, the stability of ds-FV57(VL85Ser) was notably improved, and its in vitro neutralizing potency against RV infection was enhanced. Our findings from these stabilization modifications support the feasibility of developing scFvs for PEP treatment of rabies.

Identification of binding epitope for anti-rabies virus glycoprotein single-chain Fv fragment FV57.

Single-chain Fv fragment (scFv) of anti-rabies glycoprotein (G protein) has been recommended as a new agent for detecting and neutralizing lethal rabies virus. In this study, we constructed scFv that corresponded to the FV fragment of CR57, a monoclonal antibody against rabies virus, and called it FV57. Despite its virus neutralization activity, FV57 may or may not recognize the same epitope as that recognized by CR57. To resolve this issue, the binding epitope of rabies virus G protein recognized by FV57 was identified. A recombinant rabies virus G protein fragment (RVG179; residues 179-281) comprising several epitopes was expressed in E.coli, purified, and the specificity of its binding with FV57 was determined. In addition, a peptide (abbreviated as EP, residues 224-236) comprising the known epitope of G protein to which CR57 binds was synthesized and the potency of its binding with FV57 was also determined. The results showed that FV57 could specifically bind to RVG179 and EP. Competitive ELISA experiments indicated that RVG179 and EP were able to compete with the rabies virus G protein for binding with FV57. Since no other epitope within residues 224- 236 has been reported, except for the epitope to which CR57 binds (residues 226-231), the epitope recognized by FV57 was the same as its intact antibody CR57. This demonstrated that the complementarity-determining regions (CDRs) of the heavy and light chains of FV57 have folded into the correct conformation as those of CR57.

[Studies on antigencity of human immunodeficiency virus type 1 (HIV-1) external glycoprotein as well as its expression in Pichia pastoris].

Based on the computer simulation, we analyzed hydrophobicity, potential epitope of recombined subtypes HIV-1 Env protein (851 amino acids) from Guangxi in China. Compared with conservative peptides of other subtypes in env protein, three sequences (469-511aa, 538-674aa, 700-734aa) were selected to recombine into a chimeric gene that codes three conservative epitope peptides with stronger antigencity, and was constructed in the yeast expression plasmid pPICZB. Chimeric proteins were expressed in Pichia pastoris under the induction of methanol, and were analyzed by SDS-PAGE and Westernblot. The results showed that fusion proteins of three-segment antigen were expressed in Pichia pastoris and that specific protein band at the site of 40kD was target protein, which is interacted with HIV-1 serum. The target proteins were purified by metal Ni-sepharose 4B, and were demonstrated to possess good antigenic specificity from the data of ELISA. This chimeric antigen may be used as research and developed into HIV diagnostic reagents.

HPV prevalence, E6 sequence variation and physical state of HPV16 isolates from patients with cervical cancer in Sichuan, China.

OBJECTIVES: Infection with high-risk human papillomavirus (hr-HPV) is an important factor associated with cervical cancer. The genetic mutation of HPV16 E6 and integration of HPV16 DNA in the cervical carcinoma tissues are considered important genetic changes in cervical lesion progression. But the studies of hr-HPV epidemiology are relatively less in the area of Sichuan, China. Therefore, we investigated the prevalence of 9 high-risk subtypes and analyzed the genetic mutation characteristic of HPV16 E6 and physical state of HPV16 DNA. METHODS: The fragments of L1 and E6 genes were amplified by PCR or nested PCR and then directly sequenced. Further, the multiplex PCR for HPV16 E2 and E6 genes was performed for detection of integration. RESULTS: HPV16, 58 and 18 were prominent, accounting for 78.6%, 20.0% and 9.7%, respectively in 145 isolates. E6 variants revealed that the European (EP) prototype and East Asia (EA) strain were 26 (23.0%) and 34 (30.1%), respectively. Furthermore, there were 14 base substitutions in E6 regions of the study group, of which 12 resulted in amino acid changes and the rest was silent mutation. Significantly, the 240G substitution exactly located the P53 degradation site. Overall, 8 of 114 (7.0%) isolates only contained integrated HPV16 DNA, 43 (37.7%) only contained episomal DNA and 63 (55.3%) contained both integrated and episomal DNA. The proportion of disruption of an intact E2 gene in the patients with cervical cancer is much lower than that in the previous studies. CONCLUSIONS: HPV16, 58 and 18 were mainly prevailing subtypes in patients with cervical cancer from Sichuan areas, China and EP/EA strains were predominant in these areas. Some mutations of E6 gene, which lead to the amino acid changes, may be more potentially carcinogenic and the proportion of disruption of an intact E2 gene is much lower.

[Genomic sequence of hepatitis A virus L-A-1 vaccine strain].

OBJECTIVE: To study the genome sequence of hepatitis A virus L-A-1 strain which has been applied for live attenuated vaccine production in China, to compare with other HAV strains, to understand some characteristics of L-A-1 strain, and to find the mechanism of attenuation and cell adaptation. METHODS: Genome fragments were prepared by antigen-capture PCR from infected cell (2BS), PCR products were cloned into T vector, sequenced and analyzed by using bioinformatics program. RESULTS: Analysis of the genomic sequences(nt 25-7,418) showed that the open reading frame contains 6,675 nucleotides in length encoding 2,225 amino acids. Sequence homology comparison showed 98.00% and 94.00% homology at nucleotide level, and 98.51% and 98.65% homology at amino acid level with international strains MBB and HM 175, respectively. Through comparison with other attenuated, cell adapted and cytopathic effect (CPE) strains, L-A-1 strain had mutation at nt 152, 591, 646, 687 and insertion at nt 180-181 in 5?NTR and had mutation at nt 3,889 (aa 1 052-Val) in 2B region, these mutations and insertion are molecular basis for cell adaptation; mutation at nt 4,185 (aa 1 152-Lys) in 2C region should be attenuated marker; deletion in 3A region (nt 5,020-5,025) that caused two amino acids deletion is virus fast growth basis. CONCLUSION: Through analyzing L-A-1 strain genomic sequence, certain sites related to cell adaptation and attenuation were found.