[Effect of Biochar Structure on Adsorption Characteristics of Ammonia Nitrogen].

Ammonia inhibition is a common phenomenon in biogas engineering which is rich in organic nitrogen substrate. Ammonia nitrogen in anaerobic digestate slurry can be fixed by biochar adsorption. Biochar is prepared from corn stalks and rice husks as raw materials at different temperatures (350℃, 450℃, and 550℃). The purpose is to explore the correlation between the physical and chemical structure of biochar and the adsorption characteristics of ammonia nitrogen. The structure and physicochemical properties of biochar were analyzed by elemental analysis, FTIR, BET, etc., and batch adsorption experiments were conducted to investigate the effect of biochar with different physicochemical properties on adsorption characteristics of ammonia nitrogen. The results indicated that the carbon and ash content in biochar increases with an increase in pyrolysis temperature; the NH4+-N adsorption of the corn stalk biochar prepared at 450℃ (CS450) and the rice husk biochar prepared at 550℃ (RH550) follows the quasi-secondary-and quasi-first-order kinetic models. The Freundlich adsorption model can better describe the isothermal adsorption process of ammonia nitrogen in CS450 and RH550 biochar. The adsorption capacity of corn straw carbon correlated strongly with its surface functional groups. The most significant correlation with the adsorption capacity of rice husk carbon is the specific surface area of biochar, followed by surface functional groups, and finally ash content. Among them, RH550 had the best adsorption performance, and the maximum adsorption capacity was 12.16 mg·g-1.

The influence of porcine epidemic diarrhea virus on pig small intestine mucosal epithelial cell function.

Porcine epidemic diarrhea (PED) is a highly contagious, acute enteric tract infectious disease of pigs (Sus domesticus) caused by porcine epidemic diarrhea virus (PEDV). PED is characterized by watery diarrhea, dehydration, weight loss, vomiting and death. PEDV damages pig intestinal epithelial tissue, causing intestinal hyperemia and atrophy of intestinal villi, with formation of intestinal epithelial cell cytoplasmic vacuoles. Since pig small intestinal epithelial cells (IECs) are target cells of PEDV infection, IEC cells were utilized as a model for studying changes in cellular activities post-PEDV infection. Monitoring of Na+-K+-ATPase and Ca2+-Mg2+-ATPase activities demonstrated that PEDV infection decreased these activities. In addition, IECs proliferation was shown to decrease after PEDV infection using an MTT assay. Moreover, IECs apoptosis detected by flow cytometry with propidium iodide (PI) staining was clearly shown to increase relative to the control group. Meanwhile, animal experiments indicated that PEDV virulence for IEC cells was greater than viral virulence for Vero cells, although this may be due to viral attenuation after numerous passages in the latter cell line. Collectively, these studies revealed viral pathogenic mechanisms in PEDV-infected IECs and offer a theoretical basis for PEDV prevention and control.

Euphornin reduces proliferation of human cervical adenocarcinoma HeLa cells through induction of apoptosis and G2/M cell cycle arrest.

BACKGROUND: The plant Euphorbia helioscopia L. has been used in traditional Chinese medicine for treating various disorders such as tuberculosis and edema. The aim of this study was to investigate the effect of euphornin, a bioactive compound isolated from E. helioscopia, on proliferation of human cervical adenocarcinoma HeLa cells by analyzing cell viability, rate of apoptosis, and cell cycle progression. MATERIALS AND METHODS: The sulforhodamine B assay was used to study the effect of euphornin on the proliferation of HeLa cells. Morphological changes to cell nuclei were identified after Hoechst 33342 staining. Mitochondrial membrane depolarization (MMP) was analyzed after staining with JC-1 dye. The influence of euphornin on the apoptosis rate was analyzed by Annexin V/propidium iodide double staining. Fluorescence-activated cell sorting was applied to investigate the influence of euphornin on cell cycle progression. Proteins were obtained from HeLa cells and analyzed by Western blots. RESULTS: A cell viability assay showed that euphornin inhibited proliferation of HeLa cells in a dose-dependent and time-dependent manner. Euphornin also induced apoptosis in a concentration-dependent manner, with the rates of apoptosis ranging from 25.3% to 52.6%. A high concentration of euphornin was found to block HeLa cells at the G2/M stage. A Western blot analysis suggested that euphornin might exhibit antitumor activity by inducing apoptosis. Euphornin treatment altered the ratio of Bax/Bcl-2 in HeLa cells, which led to the release of cytochrome complex. The levels of cleaved caspase-3, caspase-8, caspase-9, and caspase-10 were also markedly increased by euphornin treatment. Analysis of cell cycles indicated that euphornin induced cell cycle arrest by increasing the level of the phospho-CDK1 (Tyr15) protein. The various assays demonstrated that euphornin treatment resulted in a significant suppression of cell growth accompanied by G2/M cell cycle arrest and increased rate of apoptosis via mitochondrial and caspase pathways. CONCLUSION: Our findings suggest that euphornin has the potential to be used as a cancer therapeutic agent against human cervical adenocarcinoma.

Infection, genetic and virulence characteristics of porcine epidemic diarrhea virus in northwest China.

From September 2015 to May 2016, epidemic outbreaks of a diarrheal disease caused severe economic losses to the swine industry in northwest China. Typical clinical symptoms of the disease included severe diarrhea, vomiting, dehydration and death. In order to identify the pathogen, 27 intestinal samples were collected from dead piglets in Shaanxi, Gansu and Qinghai provinces and from Ningxia Hui Autonomous Region. All samples were tested using RT-PCR to detect rotavirus (RV), porcine epidemic diarrhea virus (PEDV) and transmissible gastroenteritis virus (TGEV). Twenty-five fecal samples were PEDV positive and five were TGEV positive; no piglets were infected with RV, indicating PEDV was the major pathogenic agent of viral diarrheal disease in these areas. Six strains were successfully isolated from positive samples and were serially passaged 40 times in Vero cells, with obvious cytopathic effects observed after 24 h post inoculation (hpi) and virus titers reaching 1.0 × 107 to 5.62 × 108. Sequence analysis ruled out that isolated strains were vaccine PEDV strains or strains derived from vaccine strains. Five strains belonged to classical strains, while one strain was a novel variant strain. The virulence of new novel variant strain SX1 and classical strain NX1 were tested in vivo using 10-day-old nursing piglets, revealing that both strains were highly pathogenic for piglets with destruction to small intestinal villi. Hematoxylin and eosin staining demonstrated markedly increased mucosal thickness, reduced villus length and villus/crypt (V/C) ratio in infected piglets. These pathological changes correlated with observed significantly reduced intestinal digestion and absorption functions that led to anorexia, dehydration, diarrhea and emaciation. Collectively, this study first reported the PEDV epidemic and phylogenetic analysis in northwest China and the results were important to understanding the infectivity, genetic characteristics, evolution and pathogenicity of PEDV strains, therefore, this experiment had important public health significance.

Efficacy of ultrasound and nerve stimulation guidance in peripheral nerve block: A systematic review and meta-analysis.

Evidence was controversial about whether nerve stimulation (NS) can optimize ultrasound guidance (US)-guided nerve blockade for peripheral nerve block. This review aims to explore the effects of the two combined techniques. We searched EMBASE (from 1974 to March 2015), PubMed (from 1966 to Mar 2015), Medline (from 1966 to Mar 2015), the Cochrane Central Register of Controlled Trials and clinicaltrials.gov. Finally, 15 randomized trials were included into analysis involving 1,019 lower limb and 696 upper limb surgery cases. Meta-analysis indicated that, compared with US alone, USNS combination had favorable effects on overall block success rate (risk ratio [RR] 1.17; confidence interval [CI] 1.05 to 1.30, P = 0.004), sensory block success rate (RR 1.56; CI 1.29 to 1.89, P < 0.00001), and block onset time (mean difference [MD] -3.84; CI -5.59 to -2.08, P < 0.0001). USNS guidance had a longer procedure time in both upper and lower limb nerve block (MD 1.67; CI 1.32 to 2.02, P < 0.00001; MD 1.17; CI 0.95 to 1.39, P < 0.00001) and more patients with anesthesia supplementation (RR 2.5; CI 1.02 to 6.13, P = 0.05). USNS guidance trends to result in a shorter block onset time than US alone as well as higher block success rate, but no statistical difference was demonstrated, as more data are required. © 2017 IUBMB Life, 69(9):720-734, 2017.

[Progress on molecular biology of Isaria farinosa, pathogen of host of Ophiocordyceps sinensis during the artificial culture].

Isaria farinosa is the pathogen of the host of Ophiocordyceps sinensis. The present research has analyzed the progress on the molecular biology according to the bibliometrics, the sequences (including the gene sequences) of I. farinosa in the NCBI. The results indicated that different country had published different number of the papers, and had landed different kinds and different number of the sequences (including the gene sequences). China had published the most number of the papers, and had landed the most number of the sequences (including the gene sequences). America had landed the most numbers of the function genes. The main content about the pathogen study was focus on the biological controlling. The main content about the molecular study concentrated on the phylogenies classification. In recent years some protease genes and chitinase genes had been researched. With the increase of the effect on the healthy of O. sinensis, and the whole sequence and more and more pharmacological activities of I. farinosa being made known to the public, the study on the molecular biology of the I. farinosa would be deeper and wider.

[Effect of wound to growth of larva of host to Ophiocordyceps sinensis during artificial breeding].

To clear the effect of the wound to the growth of the larva of the host to the Ophiocordyceps sinensis, the wounds of same severity at the same position were made artificially to the larva and which were artificial fed at the same environment and condition. The results indicated that, over the winter, the survival rate of the wounded of the infection larva was lower than that of the healthy larva, but the weight had no significant difference between the wounded and the healthy larva. The survival rate of the wounded of the no infection larva was lower than that of the healthy larva, but except with black skin, the wounded larva with offwhite and dusty red had no influence on the variety of the weight. In summery, wound had no advantage to the survival rate, but had no influence to the weight. The result had provided theoretical basis to the reforming of the system of the artificial culture O. sinensis.

[Cloning, eukaryotic expression and spatial expression patterns of porcine MNSFbeta].

MNSFbeta (Monoclonal nonspecific suppressor factor beta) is a natural immunosuppressive factor which has been reported to be involved in various biological processes, such as immune responses, cell division, stress response, cell apoptosis, and nuclear transport. However, study on porcine MNSFbeta has been rarely reported. In this study, the full-length sequence of porcine MNSFbeta (GenBank accession number: KF77642500) was predicted in silicon and its cDNA sequence was obtained through RT-PCR from porcine spleen. The nucleic acid and protein sequences were analyzed. Then, the gene was subcloned into pEGFP-C1 to construct a recombinant plasmid pEGFP-MNSFbeta which was transfected into swine umbilical vein endothelial cells (SUVECs) using Lipofectamine 2000. The expression of GFP was detected by fluorescence microscopy, Western blot, and laser confocal fluorescence microscopy. The spatial expression patterns of porcine MNSFbeta were detected by real-time qPCR. Results showed that the full length of porcine MNSFbeta was 402 bp encoding 133 amino acids with only one exon. Bioinformatics analysis showed that porcine MNSFbeta protein was a stable protein consisting of a ubiquitin-like domain fused to the ribosomal protein S30 with no signal peptide. The analyses of homology and phylogenetic tree of porcine MNSFbeta and its homologs in other 18 species showed that the identities of MNSFbeta protein sequence were higher than 91% among different species and the evolutionary distance was less than 0.05. It indicates that MNSFbeta is highly conserved in the process of evolution. Fluorescence signal showed that the fusion protein GFP-MNSFbeta was successfully expressed in SUVECs which was then confirmed by Western blot. Laser confocal fluorescence microscopy showed that MNSFbeta was expressed in both nucleus and cytoplasm. Analysis of spatial expression patterns showed that procine MNSFbeta was widely expressed in immune tissues, but not in lung, suggesting that MNSFbeta may play an important role in immune response.

[Biological activity of purificated swine XCL1 protein and preparation of its polyclonal antibody].

AIM: To obtain purificated his-XCL1 recombinant protein of porcine in vitro and prepare the associated polyclonal antibody; To study how the recombinant protein affects on lymphocytes proliferation. METHODS: Purify the recombinant protein using HiTrap(TM); Chelating HP chromatographic column; Check the purified product using SDS-PAGE; Detect the expression of the recombinant protein using Western blot; Immunize the experimental animals with the purified fusion protein to prepare the serum containing the associated polyclonal antibody. The serum will then undergo double immnodiffusion test and indirect ELISA test to determine the polyclonal antibody titer. Then, test the condition of lymphocytes proliferation by MTT. RESULTS: Single target strip could be seen under conducting the SDS-PAGE electrophoresis when the concentration of the binding buffer is 40 mmol/L and the concentration of the elution buffer is 500 mmol/L; Western blot test showed that the recombinant protein could be successfully expressed; Double immunodiffusion test showed the antigen-antibody binding ratio to be 1:8, and the titre of antibodies was 1:12 800 detected by indirect ELISA. The result of MTT showed that both the native XCL1 and the recombinant protein could stimulate lymphocytes proliferation, and this stimulating effect could be effectively blocked by the polyclonal antibody we prepared. CONCLUSION: To conclude, this recombinant protein has biological activity and this research can provide basic material for further investigation of the function of XCL1 in swine.

[Studies on the chemical constituents from the leave of Paulownia tomentosa].

OBJECTIVE: To study the chemical constituents of the leave of Paulownia tomentosa. METHODS: The constituents were isolated by column chromatography and their structures were elucidated through spectroscopic analysis. RESULTS: The compounds were identified as apigenin(I), uteolin(II), homoeriodictyol(III), 3alpha-hydroxyurs-12-en-28-oic acid (IV),3beta,19alpha-dihydroxyurs-12en-28-oic acid (pomolic acid)(V),2alpha,3a-dihydroxyurs-12-en-28-oic acid(VI),ursolic acid (VII),2alpha,3beta-dihydroxyolean-12-en-28-oic acid (maslinic acid) (VIII), daucosterol(IX),beta-sitosterol(X). CONCLUSION: The compound I-X are obtained from the leave of Paulownia tomentosa (Thunb.) Steud for the first time.

[Cloning, prokaryotic expression of novel swine gene P58IPK and its polyclonal antibody preparation].

AIM: To clone a novel swine gene P58(IPK)[58-kDa(inhibitor of protein kinase) protein] and prepare its polyclonal antibody for further research of influenza and host interaction. METHODS: The swine P58(IPK); gene was first identified in silico through homology searching in the swine EST database. Then this gene was amplified by reverse transcription polymerase chain reaction (RT-PCR). The cDNA of the gene contained the complete open reading frame(ORF) of 1 518 bp, and encoded 505 amino acid residues (Accession No.HQ287801). The gene was first analyzed using bioinformatics methods. Then P58(IPK) was cloned into a prokaryotic expression vector pET-32a to construct a recombinant plasmid named as pET-P58(IPK). The fusion protein his-P58(IPK) was expressed in E.coli BL21 and purified using a his-tag protein purification column. Subsequently rabbits were immunized with the purified protein. RESULTS: Specific polyclonal antibody against the fusion protein his-P58(IPK) was obtained. The activity of the antibody was determined through double-immunodiffusion test. The titer of the antibody was 1:20 000 as shown by ELISA. specifically recognized the protein P58(IPK) by Western blot and immunofluorescence assay. CONCLUSION: The novel swine gene P58(IPK) has been successfully cloned and its polyclonal antibody has been prepared.

[Cloning and expression of novel swine gene BCL-G(L) in E.coli and preparation of its polyclonal antibody in guinea pigs].

AIM: In order to express a novel gene named as BCL-G(L); of swine in E.coli and prepare its polyclonal antibody. METHODS: The contig sequence of the gene was predicted and in silicon cloned by blasting the human BCL-G(L); in swine ESTs database in NCBI. The cloning sequence was obtained by RT-PCR from swine spleen. The cloning sequence was identified by sequencing and compared with the contig sequence. Then the gene was cloned into a prokaryotic expression vector pET-32a to construct a recombinant plasmid named as pET32a-BCL-G(L);. The fusion protein pET32a-BCL-G(L); was expressed in E.coli BL21 and purified using a His-tag fusion protein purification kit. Then guinea pigs were immunized with the purified protein to get the specific polyclonal antibody. RESULTS: The titer of the antibody was 1:800 detected by ELISA. The protein BCL-G(L); can be specifically detected by western blot assay using the polyclonal antibody. CONCLUSION: The novel swine gene BCL-G(L); was cloned and expressed in E.coli and its polyclonal antibody was prepared successfully.

[Studies on the chemical constituents from flower of Paulownia fortunei].

OBJECTIVE: To study the chemical constituents of the flower of Paulownia fortunei. METHODS: The constituents were isolated by column chromatography and their structures were elucidated through spectroscopic analysis. RESULTS: The compounds were identified as: apigenin (I), luteolin (II), Hesperetin (III), Naringenin-7-O-beta-D-glucoside (IV), Arbutin (V), 4-hydroxybenzyl-beta-D-glucoside (VI), abscisic acid (VII) and 1-acetoxy-3-hydroxypropan-2-yl-3-hydroxypentanoate (VIII). CONCLUSION: All these compounds are obtained from the flower of this plant for the first time.

[In silico identification, molecular cloning and verification of a novel pig gene homologous to human BCL10 of innate immunity and its preliminary expression profiles in pigs].

We identified and characterized a novel swine gene Bcl10 with GenBank (Accession No. EU088132) which was homologous to human BCL10 (B-cell CLL/lymphoma 10) gene. The full-length cDNA of 925 bp for swine BCL10 was in silico cloned, and then its ORF of 702 bp coding 233 amino acid residues was verified by RT-PCR and DNA sequencing. NCBI BLAST assay indicated that this cDNA contained three extrons with a length of 57, 289 and 356 bp respectively, and it was located on the chromosome 4 of pig genome. Using semi-quantitative PCR, preliminary expression profiles of swine BCL10 were verified in different swine tissues. The expression of swine BCL10 was verified by GFP marker and RT-PCR assay. We found that, BCL10 expressed in high level in swine spleen, and with a modest level in thymus, brain and lymph node; low level mRNA was expressed in liver and not detectable level in kidney. The swine BCL10 gene was cloned to the GFP-containing eukaryotic expression vector pEGFP-C1 and transfected to PK-15 cell line by lipofectin. BCL10 was ex-pressed effectively in PK-15 cells. In summary, we cloned a novel swine BCL10 gene, and investigated its expression char-acters. This will be the fundament of the future research on its function.

[Effects of slow release-fertilizers on yield, nutrient contents and quality of Coptis chinensis].

OBJECTIVE: To study the effects of four kind of slow-release fertilizers on yield and quality of Coptis chinensis. METHOD: One to three years C. chinensis was fertilized with slow-release fertilizers twice in April and in September. The yield and nutrient content along with quality of C. chinensis were measured after two years growth. RESULT AND CONCLUSION: All of the slow-release fertilizers increased the yield obviously, and the effect of SRF1 and SRF4 is the most significant. Comparing with control group, the N content in aerial part of 1-2 year-old C. chinensis treated with SRF1 and SRF4 was lower and P and K were higher than that of control group, and the N content in aerial part of 3 year-old C. chinensis treated with SRF1 and SRF4 was higher and P and K were higher than that of control group; The N content in the root of land 3 year-old C. chinensis treated with SRF1 and SRF4 showed no significant difference comparing with control group, and P and K were lower than that of control group, the N and P content in root of 2 year-old C. chinensis treated with SRF1 and SRF4 was higher and K were lower than that of control group. After two years growth berberine content of C. chinensis treated with SRF1, SRF2 and SRF3 were significantly increased, and total alkaloid content of C. chinensis treated with SRF1, SRF3 and SRF4 were significantly increased. We recommend that SRF4 is used as the special fertilizer for 1-year-old C. chinensis, and the SRF1 and SRF4 for 2-year-old C. chinensis, and the SRF1 for 3-year-old C. chinensis.

[Effects of integrated pest control techniques to growth of host larvae Cordyceps sinensis].

UNLABELLED: To study the effects of the integrated pest control techniques on growth of host larvae of Cordyceps sinensis. METHOD: The integrated pest control techniques were compared with conventional techniques to evaluate the effects on growth of host larvae. RESULT AND CONCLUSION: The results showed that the techniques had broken the balance of the microbial living in the material, produced effective inhibition on the pests, raised the survival rate and promoted the growth of the host larvae at the same time.

[Diagnosis of coronary artery disease with 99Tcm-N-NOET myocardial perfusion imaging].

OBJECTIVE: To evaluate the sensitivity and specificity of (99)Tc(m)-N-NOET myocardial perfusion tomographic imaging (MPI) for the diagnosis of coronary artery disease (CAD). METHODS: Coronary angiography and (99)Tc(m)-N-NOET myocardial perfusion tomographic imaging were performed in the patients hospitalized in the Department of Cardiology, Peking Union Medical College Hospital, from June to December 2005 with known or suspected diagnosis of CAD. Adenosine was infused intravenously at a rate of 140 microg x kg(-1)min(-1) for 6 minutes. At the third minute of adenosine infusion, 740 MBq (20 mCi) (99)Tc(m)-N-NOET was injected intravenously. MPI was obtained 15 minutes after the (99)Tc(m)-N-NOET infusion. If the result was abnormal, rest myocardial perfusion imaging would be performed 2 hours later. Coronary angiography was performed in all patients within one week after the myocardial perfusion imaging. RESULTS: Myocardial perfusion imaging was performed in 53 cases, 36 males and 17 females, aged 56 +/- 10, who underwent coronary angiography, among which 31 had > or = 50% stenosis of coronary artery and 23 had normal coronary artery. All of the patients with stenosis of coronary artery had positive (99)Tc(m)-N-NOET adenosine stress myocardial perfusion imaging. Nineteen out of the 29 cases without stenosis of coronary artery had negative adenosine myocardial perfusion imaging. The sensitivity and specificity of (99)Tc(m)-N-NOET adenosine myocardial perfusion imaging were 100% and 73% respectively in the detection of stenosis of coronary arteries. Seven cases got percutaneous coronary intervention and 2 got coronary artery bypass graft. Six of the 9 patients undergoing revascularization had stenosis of stents or grafts, 5 of which had positive myocardial perfusion imaging. 3 cases hadn't restenosis and their results of myocardial perfusion imaging were negative. CONCLUSION: (99)Tc(m)-N-NOET myocardial perfusion imaging is a useful non-interventional method for detecting coronary artery stenosis.

[Clinical significance of adenosine 99mTc-MIBI myocardial perfusion single photon emission computed tomography for patients in percutaneous coronary intervention].

OBJECTIVE: To analyze the clinical significance of adenosine (99m)Tc-MIBI myocardial perfusion single photon emission computed tomography (SPECT) in patients with coronary artery disease (CAD) for percutaneous coronary intervention (PCI). METHODS: Coronary angiography and adenosine (99m)Tc-MIBI myocardial perfusion SPECT were performed for all patients. Adenosine myocardial perfusion was performed after PCI. Adenosine was infused intravenously at a rate of 140 microg.kg(-1).min(-1) for 6 minutes, and 925MBq (99m)Tc-MIBI was injected intravenously at 3 minutes after adenosine infusion. SPECT myocardial imaging acquisition was obtained in 1.5 hours after adenosine infusion. If the result was abnormal, rest (99m)Tc-MIBI myocardial perfusion SPECT would be performed next day. There were 17 segments of left ventricle, and four degrees of myocardial perfusion. RESULTS: There were 63 cases (63 +/- 10 years old) with CAD, in which 40 patients got PCI. Twenty eight cases after PCI. CONCLUSION: Adenosine myocardial perfusion imaging will be useful in detecting regional myocardial perfusion abnormalities for patients with PCI.

[Correction of five different types of errors of model REFSEQs appeared in NCBI human gene database only by using two novel human genes C17orf32 and ZNF362].

Found that there exist many mistakes in the REFSEQ issued in the genome annotation project of NCBI, the result of which indicates that people be cautious in using REFSEQ database in NCBI. By adopting the technical route combining bioinformatics analysis and experimental verification, through the comparison of the cloned genes in the non-redundant database, we found that there were many mistakes in the computer annotation human genome coding sequences that were issued on the internet. First we quoted nine wrong types of novel human genes anticipated by NCBI GENOME Annotation Project. Here we give one example in detail: (1) Comparison of the sequences between novel human gene C17orf32 and hypothetical human gene LOC124919. LOC123722 is a modified sequence of C17orf32 cDNA with an inserted G between 406 -407 nucleotides. The base G in the 401 position of LOC123722 cDNA is a redundant insert, which causes a reading frame shift in the translation of an alternative protein. This inserted G has not been found in our experimental clone, and is fully rejected by human EST alignment, and is shown as a redundance by genomic GT/AG organization analysis. (2) Comparison of the sequences between novel human gene C17orf32 and hypothetical human gene LOC147007. C17orf32 gene (ORF from 31 to 657 nucleotides) is located on human chromosome 17(Accession No. NT_010808.7), and is only linked with a hypothetical human gene LOC147007 (ORF from 55 to 435 nucleotides) at present. This hypothetical human gene sequence has not been verified by experiment, and is a wrong form of our verified C17orf32 gene. The full-length 1 679 bp cDNA sequence of C17orf32 exhibits overall homology to that of LOC147007 of 625 bp mRNA, with matching percentage of 37% in 36% of total window over the full-length nucleotide, especially 121 approximately 366 bp of LOC147007 is just the same as 316 approximately 561 bp of C17orf32. Thus, the 126 aa protein encoded by XP_097165 of LOC147007 exhibits overall homology to the 208 aa protein encoded by C17orf32, with matching percentage of 50% in 48% of total window over the full-length protein, especially 23 approximately 104 aa of XP_097165 is just the same as 96 approximately 177 aa of C17orf32 protein. Both flanking regions of LOC147007 outside the same ORF central part are wrong assembly of non-relative cDNA. In addition, we have in silico cloned a novel mouse gene, ORF32 (open reading frame 32) with TPA accession number of BK000258, which is the mouse ortholog of human C17orf32. Our strategy is helpful in both finding out more novel human genes and correcting the mistakes in the REFSEQs issued by NCBI genome annnotation project. For example, we adopted the gene anticipating method, through automatic calculation and analysis, anticipated two modes reference sequences (LOC124919 and LOC147007) from NCBI contig NT_ 010808. Both of them should be C17orf32, but the fact is that both of them are various wrong forms of C17orf32, respectively are the first type and second type of mistakes. Another example, we adopted gene anticipation method, through automatic calculation and analysis, anticipated three modes reference sequences (LOC14907, LOC200084 and LOC91126) from NCBI contig NT_004511 which really are one type of gene of ZNF362, but submitted three different wrong forms of ZNF362, respectively are: the fourth, fifth, and seventh type of mistakes. We can correct or avoid the currently wrong human genome coding sequence by using in silico clone and combining experimental verification. People should be cautious in treating the computer's annotation which may exist all type of wrong human genome coding sequences. The correct identification and annotation of the novel human genes still remain to be a long and arduous task.

[Analysis, identification and correction of some errors of model refseqs appeared in NCBI Human Gene Database by in silico cloning and experimental verification of novel human genes].

We found that human genome coding regions annotated by computers have different kinds of many errors in public domain through homologous BLAST of our cloned genes in non-redundant (nr) database, including insertions, deletions or mutations of one base pair or a segment in sequences at the cDNA level, or different permutation and combination of these errors. Basically, we use the three means for validating and identifying some errors of the model genes appeared in NCBI GENOME ANNOTATION PROJECT REFSEQS: (I) Evaluating the support degree of human EST clustering and draft human genome BLAST. (2) Preparation of chromosomal mapping of our verified genes and analysis of genomic organization of the genes. All of the exon/intron boundaries should be consistent with the GT/AG rule, and consensuses surrounding the splice boundaries should be found as well. (3) Experimental verification by RT-PCR of the in silico cloning genes and further by cDNA sequencing. And then we use the three means as reference: (1) Web searching or in silico cloning of the genes of different species, especially mouse and rat homologous genes, and thus judging the gene existence by ontology. (2) By using the released genes in public domain as standard, which should be highly homologous to our verified genes, especially the released human genes appeared in NCBI GENOME ANNOTATION PROJECT REFSEQS, we try to clone each a highly homologous complete gene similar to the released genes in public domain according to the strategy we developed in this paper. If we can not get it, our verified gene may be correct and the released gene in public domain may be wrong. (3) To find more evidence, we verified our cloned genes by RT-PCR or hybrid technique. Here we list some errors we found from NCBI GENOME ANNOTATION PROJECT REFSEQs: (1) Insert a base in the ORF by mistake which causes the frame shift of the coding amino acid. In detail, abase in the ORF of a gene is a redundant insertion, which causes a reading frame shift in the translation of an alternative protein, such as LOC124919 is wrong form of C17 orf32 (with mouse and rat orthologs determined by us). (2) Put together by mistake (with force). This is a wrong assembly of non-relating cDNA segment, such as LOC147007 is wrong form of C17orf32. (3) Mistakenly insert a base or one section of cDNA in the ORF which causes it ending beforehand, only coding cDNA sequence of N-terminal amino acids, incomplete. For example, LOC123722 is wrong form of SPRYD1, and even the human hypothetical gene LOC126250 or PDCD5 is wrong form of our PDCD5 (TFAR19). (4) Incomplete, only coding cDNA sequence of C-terminal amino acids. For example, human LOC149076 and mouse LOC230761 are wrong form of our verified human ZNF362 and mouse Zfp362, respectively. (5) Incomplete, only coding one section of coding protein cDNA sequence of correct gene ORF, lacking N-terminal and C-terminal amino acids sequence, and at the same time, mistakenly anticipates the first non-initiation codon amino acid of the incomplete protein amino acid as the initiation codon, e.g. anticipating L as M. For example, LOC200084 is wrong form of ZNF362. (6) Mistakenly insert a base or one section of cDNA in the ORF, wrongly causing unwanted termination codon before the insertion, so the coding protein lacks the first part of the amino acids. For example, the GenBank Acc. No. AL096883 ( LOCUS No. HS323M22B) is wrong form of an experimentally verified human NM_012263 with mouse ortholog of BC010510 determined. (7) It may regard the polluted genomic sequence as complete gene cDNA sequence and anticipate the so-called single exon gene, even the real one, only a small ORF in the very long single exon mRNA, while there really exists termination code in the same phase of the upper part of the ORF initiation code, no other characters accord with the gene's condition. For example, LOC91126 is wrong form of ZNF362. (8) The anticipated genes only have ORF which has no EST proofs on both terminal sides. Depending on this ORF, a complete gene cDNA with double support of EST and human genome (there are termination codes at the same phase of the upper part of ORF) which indicates the anticipated ORF reference sequence may be incorrect. For example, LOC164395 may be wrong form of novel human gene bankit4590055. (9) A similar but smaller protein-coding gene is anticipated in the range of the human genome sequence that has the support of EST experimental proof, so other new anticipated gene may be incorrect. For example, LOC167563 may be wrong form of CMYA5. However,these errors can be corrected or avoided by using our strategy. Here we give one example in detail: Comparision of the sequence SPRYD1 with human hypothetical gene LOC123722. The TAA bases in the position of 478-480 in LOC123722 cDNA is redundant, which causes a reading frame shift in the translation of an alternative protein. The redundancy of GTAAA of LOC123722 is not supported by our experimental clone,and is almost fully rejected by human EST alignment, and is shown as the next intron sequence by genomic GT/AG organization analysis. The verification of cDNA or genomic DNA sequence of SPRYD1 implies that LOC123722 has a wrong stop codon within its ORF because of the prediction program, thus being not complete cds. To sum up, by combining bioinformatics analyses with experimental verification, we have found that there are many errors of at least nine kinds appeared in NCBI GENOME ANNOTATION PROJECT REFSEQs through BLAST of our cloned genes in non-redundant database, and our strategy is helpful in correcting them, such as LOC14907, LOC200084 and LOC91126 (all of them should be ZNF362, but are three different kinds of wrong forms of ZNF362), three model reference sequences predicted from NCBI contig NT_004511 by automated computational analysis using gene prediction method, or such as LOC124919 and LOC147007 (both should be C17orf32, but are two different kinds of wrong forms of C17orf32), two model reference sequences predicted from NCBI contig NT_010808 by automated computational analysis using gene prediction method. Therefore, the correct identification and annotation of novel human genes may be still a heavy task, which can be finished within a long period of time. So human genome coding regions annotated by computer should be used with caution. The articles published in the past did not clearly point out the existence of mistakes in the NCBI human gene mode reference sequence. At the Seventh International Human Genome Conference held in April 2002, we first published the researching result on this aspect in the communication form of Posterly insert a base or one section of cDNA in the ORF, wrongly causing unwanted termination codon before the insertion, so the coding protein lacks the first part of the amino acids. For example, the GenBank Acc. No. AL096883 ( LOCUS No. HS323M22B) is wrong form of an experimentally verified human NM_012263 with mouse ortholog of BC010510 determined. (7) It may regard the polluted genomic sequence as complete gene cDNA sequence and anticipate the so-called single exon gene, even the real one, only a small ORF in the very long single exon mRNA, while there really exists termination code in the same phase of the upper part of the ORF initiation code, no other characters accord with the gene's condition. For example, LOC91126 is wrong form of ZNF362. (8) The anticipated genes only have ORF which has no EST proofs on both terminal sides. Depending on this ORF, a complete gene cDNA with double support of EST and human genome (there are termination codes at the same phase of the upper part of ORF) which indicates the anticipated ORF reference sequence may be incorrect. For example, LOC164395 may be wrong form of novel human gene bankit4590055. (9) A similar but smaller protein-coding gene is anticipated in the range of the human genome sequence that has the support of EST experimental proof, so other new anticipated gene may be incorrect. For example, LOC167563 may be wrong form of CMYA5. However, these errors can be corrected or avoided by using our strategy. Here we give one example in detail: Comparision of the sequence SPRYD1 with human hypothetical gene LOC123722. The TAA bases in the position of 478-480 in LOC123722 cDNA is redundant, which causes a reading frame shift in the translation of an alternative protein. The redundancy of GTAAA of LOC123722 is not supported by our experimental clone, and is almost fully rejected by human EST alignment, and is shown as the next intron sequence by genomic GT/AG organization analysis. The verification of cDNA or genomic DNA sequence of SPRYD1 implies that LOC123722 has a wrong stop codon within its ORF because of the prediction program, thus being not complete cds. To sum up, by combining bioinformatics analyses with experimental verification, we have found that there are many errors of at least nine kinds appeared in NCBI GENOME ANNOTATION PROJECT REFSEQs through BLAST of our cloned genes in non-redundant database, and our strategy is helpful in correcting them, such as LOC14907, LOC200084 and LOC91126 (all of them should be ZNF362, but are three different kinds of wrong forms of ZNF362), three model reference sequences predicted from NCBI contig NT_004511 by automated computational analysis using gene prediction method, or such as LOC124919 and LOC147007 (both should be C17orf32, but are two different kinds of wrong forms of C17orf32), two model reference sequences predicted from NCBI contig NT_010808 by automated computational analysis using gene prediction method. Therefore, the correct identification and annotation of novel human genes may be still a heavy task, which can be finished within a long period of time. So human genome coding regions annotated by computer should be used with caution. (ABSTRACT TRUNCATED)