Epitope delimitation: A new method for defining epitopes of human IgG-reactive antigenic peptides based on rabbit-recognized epitope motifs.

The use of precise epitope peptides as antigens is essential for accurate serological diagnosis of viral-infected individuals, but now it remains an unsolvable problem for mapping precise B cell epitopes (BCEs) recognized by human serum. To address this challenge, we propose a novel epitope delimitation (ED) method to uncover BCEs in the delineated human IgG-reactive (HR) antigenic peptides (APs). Specifically, the method based on the rationale of similarities in humoral immune responses between mammalian species consists of a pair of elements: experimentally delineated HR-AP and rabbit-recognized (RR) BCE motif and corresponding pair of sequence alignment analysis. As a result of using the ED approach, after decoding four RR-epitomes of human papillomavirus types 16/18-E6 and E7 proteins utilizing rabbit serum against each recombinant protein and sequence alignment analysis of HR-APs and RR-BCEs, 19 fine BCEs in 17 of 22 known HR-APs were defined based on each corresponding RR-BCE motifs, including the type-specificity of each delimited BCE in homologous proteins. The test with 22 known 16/20mer HR-APs demonstrated that the ED method is effective and efficient, indicating that it can be used as an alternative method to the conventional identification of fine BCEs using overlapping 8mer peptides.

A simpler and more cost-effective peptide biosynthetic method using the truncated GST as carrier for epitope mapping.

There is a need to develop better methods for epitope mapping and/or identification of antibody-recognizing motifs. Here, we describe improved biosynthetic peptide (BSP) method using a newly developed plasmid pXXGST-3 as vector, which has a viral E7 gene in the cloning sites of pXXGST-1. It is crucial to employ pXXGST-3 instead of pXXGST-1, since it makes use of the BSP method simpler and easier to perform, and more cost-effective for epitope mapping. These merits are embodied in two aspects: i) convenient recovery of double enzyme-digested product due to the existence of 315 bp inserted between BamH I and Sal I sites, and thus greatly reducing the production of self-ligation clones, and ii) no longer requiring control protein when screening recombinant (r-) clones expressing 8/18mer peptides by running polyacrylamide gel electrophoresis. The protocol involves the following core steps: (i) design of plus and minus strands of DNA fragments encoding overlapping 8/18mer peptides; (ii) chemical synthesis of the designed DNA fragments; (iii) development of r-clones using pXXGST-3 vector expressing each 8/18mer peptide fused with truncated GST188 protein; (iv) screening r-clones by running the cell pellets from each induced clone on SDS-PAGE gel followed by sequencing of inserted DNA fragments for each verified r-clone; and (v) Western blotting with either monoclonal antibodies or polyclonal antibodies. This improved GST188-BSP method provides a powerful alternative tool for epitope mapping.

Epitomics: IgG-epitome decoding of E6, E7 and L1 proteins from oncogenic human papillomavirus type 58.

To enable rational multi-epitope vaccine and diagnostic antigen design, it is imperative to delineate complete IgG-epitome of the protein. Here, we describe results of IgG-epitome decoding of three proteins from high-risk (HR-) oncogenic human papillomavirus type 58 (HPV58). To reveal their entire epitomes, employing peptide biosynthetic approach, 30 precise linear B-cell epitopes (BCEs) were mapped on E6, E7 and L1 proteins using rabbits antisera to the respective recombinant proteins. Using sequence alignment based on BCE minimal motif, the specificity and conservativeness of each mapped BCE were delineated mainly among known HR-HPVs, including finding 3 broadly antibody cross-reactive BCEs of L1 that each covers almost all HR-HPVs. Western blots revealed that 13 of the 18 BCEs within L1-epitome were recognized by murine antisera to HPV58 virus-like particles, suggesting that these are antibody accessible BCEs. Also, a highly conserved epitope (YGD/XTL) of E6 was found to exist only in known common HR-HPVs, which could be used as the first peptide reference marker for judging HR-HPVs. Altogether, this study provides systemic and exhaustive information on linear BCEs of HR-HPV58 that will facilitate development of novel multi-epitope diagnostic reagents/chips for testing viral antibodies and 'universal' preventive HPV peptide vaccine based on L1 conserved BCEs.

Mapping of epitopes relevant for induction of acrosome reaction on human zona pellucida glycoprotein-4 using monoclonal antibodies.

PROBLEM: To decipher structural and functional aspects of human zona pellucida glycoprotein-4 (ZP4), the epitopes recognized by monoclonal antibodies (MAbs) have been mapped. METHOD OF STUDY: Recombinant human ZP4-mediated induction of acrosome reaction in human sperm was studied in the absence and presence of ZP4-specific MAbs. The epitopes of MAbs were mapped using recombinant peptides expressed in Escherichia coli. RESULTS: Monoclonal antibodies (MA-1662, MA-1671) against human ZP4 showed specific binding to ZP matrix of human eggs in an indirect immunofluorescence assay. Both the antibodies showed significant (P < 0.05) inhibition in the baculovirus-expressed recombinant ZP4-mediated acrosome reaction. MA-1671 recognized N-terminal fragment of ZP4 and minimal epitope mapped to amino acid residues 126-130 (PARDR), whereas MA-1662 reacted to C-terminal fragment and minimal epitope mapped to amino acid residues 256-260 (ENELV). CONCLUSIONS: The epitopes corresponding to both N- and C-terminal parts of human ZP4 may be relevant for its biological activity.

Mapping of minimal motifs of B-cell epitopes on human zona pellucida glycoprotein-3.

The human zona pellucida glycoprotein-3 (hZP3) by virtue of its critical role during fertilization has been proposed as a promising candidate antigen to develop a contraceptive vaccine. In this direction, it is imperative to map minimal motifs of the B cell epitopes (BCEs) so as to avoid ZP-specific oophoritogenic T cell epitopes (TCEs) in the ZP3-based immunogens. In this study, based on known results of mapping marmoset and bonnet monkey ZP3 (mstZP3 and bmZP3), two predictable epitopes(23-30 and 301-320) on hZP3 were first confirmed and five minimal motifs within four epitopes on hZP3 were defined using serum to recombinant hZP3a(22-176) or hZP3b(177-348) as well as a biosynthetic peptide strategy. These defined minimal motifs were QPLWLL(23-28) for hZP3(23-30), MQVTDD(103-108) for hZP3(93-110), EENW(178-181) for hZP3(172-190), as well as SNSWF(306-310) and EGP(313-315) for hZP3(301-320), respectively. Furthermore, the antigenicity of two peptides for hZP3(172-187) and hZP3(301-315) and specificity of the antibody response to these peptides were also evaluated, which produced high-titer antibodies in immunized animals that were capable of reacting to ZP on human oocytes, r-hZP3b(177-348) protein, as well as r-hZP3(172-190), r-hZP3(303-310), and r-hZP3(313-320) epitope peptides fused with truncated GST188 protein.

Minimal motif mapping of a known epitope on human zona pellucida protein-4 using a peptide biosynthesis strategy.

An important step in the development of a human zona pellucida (huZP) peptide vaccine is to define the minimal amino acid motif for a mapped B cell epitope peptide within huZP4. Identification of this minimal motif is necessary to remove an overlapping T cell epitope that induces a pathogenic T cell response. Here we describe motif (PLTLEL(314-319)) mapping of an 18mer B cell epitope peptide(308-325) on huZP4 protein (previously known as huZP1/ZPB protein), achieved using a set of 22 biosynthetic 8mer peptides fused with truncated glutathione S-transferase (GST) or truncated streptavidin protein, and detected using rabbit anti-porcine zona pellucida (pZP) IgG. The immunogenicity of the B cell epitope peptide was evaluated in rabbits using expressed B cell epitope peptide fused with truncated streptavidin as the antigen. This construct elicited high titer antibody to the 18mer B cell epitope peptide, with reactivity to native human ZP, the biosynthetic 18mer peptide and the 18mer peptide GST fusion protein.

[Variation of below-ground carbon sequestration under long term cultivation and grazing in the typical steppe of Nei Monggol in North China].

Human activities have changed the earth surface mostly, which caused many environment issues now. We aimed to detect the process that human activities exert on ecosystem by investigating and analyzing the changes of plant community, especially underground soil and root carbon sequestration under long-term cultivation and grazing in typical steppe area of Nei Monggol, North China. The carbon sequestration on the root and soil in three plots of typical steppe area: 26-year exclosure grassland (E26), continuous grazing grassland (LG) and 35-year cultivated tillage (LC) were measured. The carbon storage in the layer of 0-40 cm showed a trend that E26 (7 307.59 g x m(-2) and 950.32 g x m(-2)) approximately LG (7834.01 g x m(-2) and 843.43 g x m(-2)) > LC (4537.04 g x m(-2) and 277.35 g x m(-2)), occupied 88.49%, 90.28% and 94.24% of total soil-root carbon respectively. The original composition structure of plant-soil system was completely destroyed by human cultivation, and it also led to sever soil erosion as well. The sand content in soil of LC at layers 0-10 cm, 10-20 cm were increasing by 81% and 39% compared to E26. On the other hand, the root biomass of LG at 0-40 cm decreased by 71%. Cultivation resulted in significant decrease of the carbon storages in soil and root. Therefore, the present cultivation should be ceased and the optimum measurements should be taken to make the tillage restore to natural grassland condition. Continuous grazing led to the significantly changes of the above-ground vegetation characteristics such as community height, coverage and biomass. While the changes of soil organic content and root biomass was not remarkable under grazing. However, the bulk density of surface soil (0-10 cm) exhibited significant increase in LG compared to E26, which indicated that the present grazing pressure have been reaching the threshold of grassland capacity. Therefore, the present grazing pressure should be decreased properly in order to avoid more serious degradation.

[Decorative repair and reconstruction of subtotal thumb and finger defect with trimmed toe flap transfer].

OBJECTIVE: In partial loss of distal finger segment, a corresponding part of the toe tissue compound is harvested and transplanted for repair or reconstruction. This new procedure gives forth a new concept and is called decorative repair or reconstruction. METHODS: In a series of 77 patients with 88 thumb and/or finger subtotal defects in forms of lateral half, dorsal half or volar half composite tissue defects were reconstructed with lateral nail-skin flap, dorsal skin-nail flap or pulp flap taken from corresponding part of the toes. The blood circulations were re-established by anastomosing digital arteries of the toe transplants and fingers. RESULTS: In this series 75 patients with 78 fingers reconstructed are successful. The overall survival rate is 97.5%. Follow-up examinations made half to 12 years postoperatively showed the fingers are having a normal length, outward appearance and function. There are nails preserved. The pulps are full. Sweating function are present. Two-point-discrimination tests are between 4-6 mm. CONCLUSION: By decorative reconstruction of subtotal dorsal, lateral, or volar halves defect of thumb and/or fingers by transplanting corresponding part of soft tissue taken from the toe has the merit of repair of any parts of tissue loss precisely what is needed. This procedure is better than any traditional toe-to-hand transfer and realizing the exact meaning of decorative reconstruction.

[Dust storms trend in the Capital Circle of China over the past 50 years and its correlation with temperature, precipitation and wind].

The trends of number of dust storm days of the selected 11 meteorological stations from their established year to 2000 as well as their correlations with temperature, precipitation and wind are revealed. The number of dust storm days of the Capital Circle of China is distinctly variable in space and time. The numbers of dust storm days of the western area are far more than those of the eastern area. The interannual variability of number of dust storm days is remarkable. The number of dust storm days of the following 7 stations, Erlianhaote, Abaga, Xilinhaote, Fengning, Zhangjiakou, Huailai and Beijing, declined along the past decades, but those of the other four stations had no significant upward or downward trends. There is a marked seasonality of the number of dust storm days, and the maximum was in April. The correlation between number of dust storm days and number of days of mean wind velocity > 5 m/s, which is critical wind velocity to entrain sand into the air, was strongest among the three climatic factor. There were significant positive correlations between the number of dust storm days and number of days of mean wind velocity > 5 m/s in 6 stations. The second strongest climatic factor correlated with the number of dust storm days is temperature. There are significant negative correlations between the number of dust storm days and mean annual temperature, mean winter temperature, mean spring temperature in 3 or 4 stations. The correlation between the number of dust storm days and precipitation is weakest. Only one station, Zhurihe, showes significant negative correlation between the number of dust storm days and spring rainfall. There are 4 stations whose number of dust storm days don't significantly correlate with the climate. In the end, the spatial-temporal variability of dust storms and its relation with climate in the Capital Circle of China were discussed thoroughly.

Variations of plant life form diversity along the Northeast China Transect and its direct gradient analysis.

Plant life form diversity and its direct gradient analysis on a larger scale climate change gradient were tested, based on the data from Northeast China Transect platform. The results showed that the species numbers, life form richness and life form diversity were relative higher at the eastern forests and the ecotone between typical vegetation, while those on the meadow grasslands and typical steppes were lower. Although plant life forms can reflect the climate variations, life form diversity is not consistent with the major global gradient along the NECT.