静电纺丝聚乙烯醇/丝素蛋白/秦皮提取物-聚己内酯双层纳米纤维膜的制备及抗氧化性能

为制备干燥面膜,解决湿剂面膜中使用防腐剂、增稠剂等成分来延长面膜保质期对皮肤造成伤害的问题,通过顺序静电纺丝技术制备了以聚乙烯醇(PVA)、丝素蛋白(SF)负载秦皮提取物(CRF)为“亲肤层”和聚己内酯(PCL)为疏水层的双层纳米纤维膜。利用扫描电子显微镜、红外光谱分析及水接触角对纤维膜进行表征,并研究了纤维膜黏附性能、释放性能、抗氧化性能及生物相容性。结果表明,PVA/SF可以负载1%,3%和5%不同浓度的秦皮提取物,在湿润后PVA/SF/CRF-PCL纤维膜具有很强的黏附性,同时CRF可以在4min左右释放平衡,达到快速释放的效果。负载5%秦皮提取物的PVA/SF纤维膜具有良好的抗氧化性能,对DPPH自由基和ABTS自由基清除率分别为89.13%±0.42%和85.49%±0.82%,PVA/SF/CRF-PCL纤维膜溶血率低,对B16细胞、Hela细胞均无毒性,B16细胞存活率均在89%以上,显示了良好的生物相容性,可以安全应用于皮肤领域。

A novel single-base mutation in CaSGR1 confers the stay-green phenotype in pepper

The stay-green trait is of considerable importance in extending the shelf life of green pepper fruit(Capsicum annuum L.)and in enhancing the appearance of ornamental plants.The study revealed the genetic and regulatory mechanisms of the stay-green trait in pepper,which will aid in the selection of ornamental pepper varieties.In this study,a pepper mutant with stay-green fruit named TNX348 was identified from a germplasm resource bank.Two segregating populations were constructed using the stay-green mutant TNX348 and then used in bulked segregant analysis combined with RNA sequencing and linkage analyses.The causal gene of the stay-green trait was mapped to an approximately 131-kb region,and a senescence-induced chloroplast protein gene,CaSGR1(Capana01g000359),was identified as a candidate gene.Sequencing analysis revealed a G→A single-base mutation of CaSGR1 in TNX348 that led to early termination of translation.Based on the single-base mutation,a single nucleotide polymorphism(SNP)marker co-segregating with the stay-green trait was developed.Furthermore,in transcriptome analysis,expression patterns of 11 hormone transduction-related transcription factors,such as abscisic acid-insensitive(ABI),abscisic acidresponsive element-binding factor(ABF),and NAC transcription factor,were similar or opposite to that of CaSGR1.The results indicated that the transcription factors might mediate chlorophyll degradation by regulating the expression of CaSGR1.

Monitoring the in vivo siRNA release from lipid nanoparticles based on the fluorescence resonance energy transfer principle

The siRNA-loaded lipid nanoparticles have attracted much attention due to its significant gene silencing effect and successful marketization.However,the in vivo distribution and release of siRNA still cannot be effectively monitored.In this study,based on the fluorescence resonance energy transfer(FRET)principle,a fluorescence dye Cy5-modified survivin siRNA was conjugated to nanogolds(Au-DR-siRNA),which were then wrapped with lipid nanoparticles(LNPs)for monitoring the release behaviour of siRNA in vivo.The results showed that once Au-DR-siRNA was released from the LNPs and cleaved by the Dicer enzyme to produce free siRNA in cells,the fluorescence of Cy5 would change from quenched state to activated state,showing the location and time of siRNA release.Besides,the LNPs showed a significant antitumor effect by silencing the survivin gene and a CT imaging function superior to iohexol by nanogolds.Therefore,this work provided not only an effective method for monitoring the pharmacokinetic behaviour of LNP-based siRNA,but also a siRNA delivery system for treating and diagnosing tumors.

Risk factors,prognostic predictors,and nomograms for pancreatic cancer patients with initially diagnosed synchronous liver metastasis

BACKGROUND Liver metastasis(LM)remains a major cause of cancer-related death in patients with pancreatic cancer(PC)and is associated with a poor prognosis.Therefore,identifying the risk and prognostic factors in PC patients with LM(PCLM)is essential as it may aid in providing timely medical interventions to improve the prognosis of these patients.However,there are limited data on risk and prognostic factors in PCLM patients.AIM To investigate the risk and prognostic factors of PCLM and develop corresponding diagnostic and prognostic nomograms.METHODS Patients with primary PC diagnosed between 2010 and 2015 were reviewed from the Surveillance,Epidemiology,and Results Database.Risk factors were identified using multivariate logistic regression analysis to develop the diagnostic mode.The least absolute shrinkage and selection operator Cox regression model was used to determine the prognostic factors needed to develop the prognostic model.The performance of the two nomogram models was evaluated using receiver operating characteristic(ROC)curves,calibration plots,decision curve analysis(DCA),and risk subgroup classification.The Kaplan-Meier method with a logrank test was used for survival analysis.RESULTS We enrolled 33459 patients with PC in this study.Of them,11458(34.2%)patients had LM at initial diagnosis.Age at diagnosis,primary site,lymph node metastasis,pathological type,tumor size,and pathological grade were identified as independent risk factors for LM in patients with PC.Age>70 years,adenocarcinoma,poor or anaplastic differentiation,lung metastases,no surgery,and no chemotherapy were the independently associated risk factors for poor prognosis in patients with PCLM.The C-index of diagnostic and prognostic nomograms were 0.731 and 0.753,respectively.The two nomograms could accurately predict the occurrence and prognosis of patients with PCLM based on the observed analysis results of ROC curves,calibration plots,and DCA curves.The prognostic nomogram could stratify patients into prognostic groups and perform well in internal validation.CONCLUSION Our study identified the risk and prognostic factors in patients with PCLM and developed corresponding diagnostic and prognostic nomograms to help clinicians in subsequent clinical evaluation and intervention.External validation is required to confirm these results.

复合材料界面微观力学表征方法及界面细观力学模型研究进展

复合材料界面的应力传递对材料的性能有着重要的影响,对于界面相的组成、结构、使用温度及强度的研究极为重要。文中对单丝复合材料界面微观测试方法进行了归纳总结,重点介绍了微观测试方法在高温下、动态载荷加载过程中、串珠纤维表征、原位检测可视化等界面表征方面的研究现状,并介绍了细观力学模型在界面失效、界面增强机理等方面的研究进展。最后分析了目前研究中界面理论的缺失,并就未来的研究方向进行了合理性建议。

先进聚合物基复合材料超声焊接研究进展

先进聚合物基复合材料(APC)取代传统金属材料实现了航空航天结构件的轻量化,其用量已成为衡量大型飞行器技术先进性的重要标志。高效连接工艺技术是影响APC构件制造成本和使用安全的关键因素之一。文中综述了纤维增强热塑性树脂基复合材料和热固性树脂基复合材料超声焊接的研究进展;详细讨论了超声波焊接机理、焊接头结构设计及焊接工艺参数(焊接时间、焊接压力、振幅、能量导向器类型等)等对APC焊接头承载性能的影响,并总结了目前APC超声焊接领域存在的问题,指出了未来的研究方向。

不同肝素钠抗凝治疗方案在肝衰竭患者血浆置换中的比较研究

目的探讨肝衰竭患者血浆置换中安全有效的肝素钠抗凝治疗方案。方法选取2018年1月—8月河南省人民医院感染性疾病科收治的肝衰竭行血浆置换患者114例为研究对象。根据不同抗凝治疗方案分为肝素钠全量组(A组),仅采用首次剂量肝素钠组(B组),以及不采用首次剂量、采用术中持续匀速泵入肝素钠组(C组)。比较3组患者血浆置换治疗前后的白细胞(WBC)、血小板(PLT)、肌酐、尿素、天门冬氨酸氨基转移酶(AST)、丙氨酸氨基转移酶(ALT)、总胆红素(TBIL)、白蛋白(Alb)、前白蛋白(PA)、D-二聚体、二氧化碳结合力(CO2CP)、血红蛋白(Hb)、凝血酶原时间(PT)、活化部分凝血活酶时间(APTT)及纤维蛋白原(FBG),以及血滤器及管道凝血情况。结果 3组患者WBC、PLT、肌酐、尿素、AST、ALT、TBIL、Alb、PA、D-二聚体、CO2CP治疗前后的差值比较,差异无统计学意义(P>0.05);3组患者的Hb、PT、APTT及FBG治疗前后的差值比较,差异有统计学意义(P <0.05);3组患者血滤器凝血发生情况为:A组1级1例,发生率为2.5%;B组1级1例,2级1例,发生率为5.0%;C组2级2例,发生率为5.9%,3组比较,差异无统计学意义(P>0.05)。3组患者均未发生管道凝血。结论血浆置换中不采用首次剂量、持续匀速泵入小剂量肝素钠、治疗结束前30 min停止使用的抗凝方案,对肝衰竭患者凝血情况的发生及凝血功能的影响最小,且不影响患者治疗效果。

Incidence and treatment of hepatic artery complications after orthotopic liver transplantation

AIM: To investigate the incidence and treatment of hepatic artery complications after orthotopic liver transplantation.METHODS: From February 1999 to May 2002, orthotopic liver transplantations (OLT) were performed in 72 patients with end-stage liver diseases with an average age of 40.2±13.6years (ranged from 11 to 68 years), 56 were males and 16 females. The preoperative evaluation for the 72 patients was performed using duplexsonography, abdominal CT scan,and angiography of the hepatic artery. All donor grafts were perfused and preserved in University of Wisconsin solution at 4 ℃. OLT was performed with standard techniques with or without a veno-venous bypass. Reconstructions of hepatic artery were performed between the branch patches of gastroduodenal/hepatic or splenic/common hepatic artery confluence of the donors and recipients, and an end-to-end anastomosis between other arterial vessels of the donors and recipients was done. Arterial anastomosis was performed with interrupted 7-0/8-0 monofilament polypropylene suture under 3.5 x loupe magnification. Diagnosis of the complications of hepatic artery after OLT was based on the clinical presentations, ultrasound findings and arterial angiography. All patients were followed up regularly for duplex ultrasound scan after discharge.RESULTS: The overall incidence of arterial complications in 72 patients after OLTs was 1.4 % (1/72). One 3 cm pseudoaneurysm at the side of anastomotic site of hepatic artery was found by urgent arteriogram due to hemoperitoneum secondary to bile leakage after OLT.Subsequently the pseudoaneurysm was successfully embolized and the blood flow toward the donor liver in hepatic artery remained. The overall postoperative 30-day mortality rate was 8.33 %. The one-year survival rate was 83.72 % in 50 patients with benign diseases and was 71.43 % in 22 patients with malignant diseases following OLT. No death associated with complications of hepatic artery occurred.CONCLUSION: Careful preoperative evaluations and intraoperative microsurgical technique for hepatic artery reconstructions are the keys in prevention of hepatic artery complications after OLT.

Comparative Transcriptome Analysis between Cytoplasmic Male-sterile Line and Its Maintainer During the Floral Bud Development of Pepper

The male-sterile line has been largely used in the hybrid seed production of pepper, which can effectively improve the efficiency of hybrid seed production. However, the formation mechanism of male sterility in pepper remains unclear. In the present study, we compared the gene expression patterns between pepper cytoplasmic male sterile line 9704 A and its maintainer 9704 B during floral bud development using RNA sequencing technology. A total of 547 976 and 2 416 Differentially Expressed Genes(DEGs) were identified in the stage S1, S2 and S3, respectively,and more than 70% of the DEGs were down-regulated in the sterile line. Gene Ontology(GO), and Kyoto Encyclopedia of Genes and Genomes(KEGG) enrichment analyses were performed to further understand the functions of these identified DEGs. The results showed that the DEGs were mainly enriched in pathways of starch and sucrose metabolism, pentose and glucuronate interconversions. A number of genes, such as MS1, PME5, ATPB, and lots of transcription factors were found down-regulated in the sterile line, and we also identified a series of genes with large differences in expression patterns between sterile line and maintainer line. Collectively, our findings laid a foundation for further molecular breeding in pepper and provided new insights into its mechanism underlying the male sterility.

REAL-TIME THREE-DIMENSIONAL ECHOCARDIOGRAPHY FOR QUANTIFYING LEFT VENTRICULAR MASS

To test the accuracy of real-time three-dimensional echocardiography (RT3DE) imaging system for evaluating left ventricular mass (LVM) in phantom and excised canine heart. Methods Ten left ventricular (LV) wall phantoms made of two rubber-bursas, ten excised canine hearts underwent RT3DE and two-dimensional echocardiography (2DE). In RT3DE "full volume" imaging, the myocardial volume was mea-sured using 2,4,8, and 16-plane method with the analysis software of RT3DE. Mass was then calculated by multiplying the resulting myocardial volume by specific density of myocardial tissue. In 2DE the masses were measured by area-length meth-od. The true LV wall phantom mass was measured by water displacement and the canine LVM was weighed by anatomy, which served as a reference standard. We compared RT3DE or 2DE with true mass. Results In LV wall phantoms, RT3DE correlated with true masses strongly (r = 0.813-0.994) and weakly correlated between 2DE and true masses (r = 0.628). In excised canine hearts, there is an excellent correlation between RT3DE and true masses (r = 0.764-0.991), while 2DE value showed a lesser correlation (r = 0.514). There are no difference between RT-3DE and true masses (P > 0.05) but different between 2DE and true masses (P < 0.05). In different planes, there was no difference between 8-plane and 16-plane (P > 0.05) but different between 8-plane and 2, 4-plane (P < 0.05). Conclusion RT3DE can accurately quantify LVM and provide a new tool to evaluate LV function. For LVM by RT3DE, 8-plane measurement method is the best choice for accuracy and convenience.

FT-IR spectroscopic analysis of normal and cancerous tissues of esophagus

AIM: To investigate the special Fourier transform infraredspectroscopy (FT-IR) spectra in normal and cancerous tissuesof esophagus.METHODS: Twenty-seven pairs of normal and canceroustissues of esophagus were studied by using FT-IR and thespecial spectra characteristics were analyzed in differenttissues.RESULTS: Different spectra were found in normal andcancerous tissues. The peak at 1 550/cm was weak andwide in cancerous tissues but strong and high in normaltissues.The ratio of I1 647/I1 550 was 2.0 in normal tissuesand 2.36 in cancerous tissues (P＜0.05). The ratio of Ⅰ1 550/I 1 080 was 4.5 in normal tissues and 3.4 in canceroustissues (P＜0.01). The peak at 1453/cm was higher than at1 402/cm in normal tissue and lower than at 1 402/cm incancerous tissues.CONCLUSION: The results indicate that F-FIR may be used in clinical diagnosis.

Inhibition effect of Chinese herbal medicine on transcription of hepatitis C virus structural gene in vitro

AIM: To investigate the inhibitory effect of Chinese herbal medicine on the transcription of hepatitis C virus (HCV) structural gene in Hela D cells.METHODS: Hela cell line was transfected with recombinant pBK-CMV-HCV containing HCV structural gene by Lipofectamine. RT-nested-PCR and Western blot assay were used to testify the HCV gene expression in Hela cells. The Hela cells expressing HCV structural protein were named Hela D cells. Prescriptions of Xiao chaihu Decoction (XCHD),Fufang Huangqi (FFHQ) and Bingganling (BGL) wererespectively added to Hela D cells in various concentrations. Semi-quantitative RT-nested-PCR product analysis was performed according to the fluorescent density between HCV DNA band and GAPDH DNA band in gel electrophoresisafter screened. RESULTS: Recombinant pBK-CMV-HCV could correctly express the HCV structural gene in Hela D cells. After coculture of Hela D cells with three prescriptional different concentrations for 48 h respectively, the transcription of HCVgene decreased with increasing of the concentration of each prescription. The lightness ratio of HCV product bands to GAPDH product bands was 0.24, 0.10 and 0.12 in Hela D cells incubated with 0.1 g/mL of XCHD, FFHQand BGL respectively and the lightness ratio HCV product bands to GAPDH product bands was 0.75, 0.67 and 0.61respectively in the control cells. CONCLUSION: The prescriptions of XCHD, FFHQ and BGL partly inhibit the transcription of HCV structural gene inHela D cells.

SDSS J022119.84+005628.4:A Radio-Loud Narrow-Line Seyfert 1 Galaxy with Star Formation in its Nucleus

The optical spectrum of SDSS J022119.84 + 005628.4 (R. A. = 02h 21m 19.84s, Dec = 00°56' 28.4', J2000; z = 0.399785 ± 0.000558) is analyzed by multi-component profile modelling. The small flux ratio of [OⅢ]NC/Hβ (=0.78) and FWHM(HβBC) = 1778.1 ±85.9 km s-1 led us to identify SDSS J022119.84 + 005628.4 as a Narrow-line Seyfert 1 galaxy with RFe= 1.4-1.0+1.0 and intermediate radio loudness, logR = 1.93. The continuum low jump at the Balmer limit might be contributed by a starburst component, which suggests that the object is a relatively young AGN with star-formation activity in its inner region. This provides a useful observational test of the AGN evolution. The low flux ratio of the narrow components, [OⅢ]/Hβ = 1.62, and the prominent [OⅡ]γ3727 emission, [OⅡ]/[OⅢ] = 2.24, suggests that there should be a group of AGNs different from Seyfert 2s when their broad line regions are obscured by the torus on the line of sight. A possible orientation effect is suggested to interpret the observed Seyfert 1.5s-like spectrum of the object. The spectrum features prominent broad blue wings of both [OⅢ]γγ4959 and 5007. After excluding other possible contributions, we conclude that the broad components are emitted by [O Ⅲ] itself. The material emitting the broad [O III] line is probably situated in a transient line region (with a low electron density, ne < 107cm-3) between the broad line region (BLR) and the narrow line region (NLR). The object can be classified as a 'blue outlier' defined by Zamanov et al., according to its velocity shift vr([OⅢ]) = -293.6 ± 18.0 km s-1, relative to the systematic velocity.

SPECIES DIVERSITY AND COMMUNITY STRUCTURE OF SUCKING LICE IN YUNNAN, CHINA

On the basis of investigating 9 counties (towns) in Yunnan Province of China, the species diversity and community structure of sucking lice on the body surface of small mammal hosts are studied in the paper. Species richness (S) is used to stand for the species diversity. The calculation of community diversity index and evenness are based on Shannon-Wiener's method. 2745 small mammals captured from the investigated sites belong to 10 families, 25 genera and 41 species in 5 orders (Rodentia, Insectivora, Scandentia, Logomorpha and Carnivora) while 18165 individuals of sucking lice collected from the body surface of the small mammal hosts are identified into 4 families, 6 genera and 22 species. The species of sucking lice are much less than the species of their hosts. Most species of small mammals have their fixed sucking lice on their body surface. One species of small mammals usually have few species of sucking lice (1 to 4 species). The close species of the hosts in the taxonomy are found to have the same or similar dominant species of sucking lice on their body surface. The results reveal that the species diversity of sucking lice on small mammals is very low with a very simple community structure. The results also imply there may be a close co-evolution relationship between the lice and the hosts.

The Structural Characterization and Antigenicity of the S Protein of SARS-CoV

The corona-like spikes or peplomers on the surface of the virion under electronic microscope are the most striking features of coronaviruses. The S (spike) protein is the largest structural protein, with 1,255 amino acids, in the viral genome. Its structure can be divided into three regions: a long N-terminal region in the exterior, a characteristic transmembrane (TM) region, and a short C-terminus in the interior of a virion. We detected fifteen substitutions of nucleotides by comparisons with the seventeen published SARS-CoV genome sequences, eight (53.3%) of which are non-synonymous mutations leading to amino acid alternations with predicted physiochemical changes. The possible antigenic determinants of the S protein are predicted, and the result is confirmed by ELISA (enzyme-linked immunosorbent assay) with synthesized peptides. Another profound finding is that three disulfide bonds are defined at the C-terminus with the N-terminus of the E (envelope) protein, based on the typical sequence and positions, thus establishing the structural connection with these two important structural proteins, if confirmed. Phyloge-netic analysis reveals several conserved regions that might be potent drug targets.

Novel Constructs of Tuberculosis Gene Vaccine and Its Immune Effect on Mice

A novel tuberculosis(TB)gene vaccine containing mouse granulocyte macrophage-colony stimulating factor (mGM-CSF)and a TB antigen(Ag85A)was developed in this study.The genes encoding Ag85A and mGM-CSF were amplified by PCR respectively from the Ag85A-containing pBSby5 and pC-mGM-CSF.The genes were then cloned into two different polylinker sites of plasmid pIRES,forming a novel TB gene vaccine construct pI85AGM. Following transfection of pI85AGM plasmid into 7721 cell line by Lipofectamine^(TM),the expression of Ag85A and GM-CSF proteins was identified by Western blotting or RT-PCR.Then Balb/c mice were inoculated with the recombinant pI85AGM,pI85A,pIGM or plasmid alone,respectively.The activities of CTL,NK cells and the Ag85A-stimulated proliferation of spleen cells were measured by MTT method.The serum antibody against Ag85A was detected by ELISA.The results showed that the Ag85A and GM-CSF proteins could be expressed in 7721 cell line and the activity of CTLs and the proliferation of spleen cells were significantly increased in the pI85AGM-immunized mice,indicating that the pI85AGM-immunized mice could generate specific immune responses to Ag85A.This study might provide possibility for developing novel anti-TB gene vaccine.Cellular & Molecular Immunology.2005;2(1):57-62.Cellular & Molecular Immunology.2005;2(1):57-62.

The E Protein Is a Multifunctional Membrane Protein of SARS-CoV

The E (envelope) protein is the smallest structural protein in all coronaviruses and is the only viral structural protein in which no variation has been detected. We conducted genome sequencing and phylogenetic analyses of SARS-CoV. Based on genome sequencing, we predicted the E protein is a transmembrane (TM) protein characterized by a TM region with strong hydrophobicity and α-helix conformation. We identified a segment (NH2-_L-Cys-A-Y-Cys-Cys-N_-COOH) in the carboxyl-terminal region of the E protein that appears to form three disulfide bonds with another segment of corresponding cysteines in the carboxyl-terminus of the S (spike) protein. These bonds point to a possible structural association between the E and S proteins. Our phylogenetic analyses of the E protein sequences in all published coronaviruses place SARS-CoV in an independent group in Coronaviridae and suggest a non-human animal origin.

Complete Genome Sequences of the SARS-CoV: the BJ Group (Isolates BJ01-BJ04)

Beijing has been one of the epicenters attacked most severely by the SARS-CoV (severe acute respiratory syndrome-associated coronavirus) since the first patient was diagnosed in one of the city's hospitals. We now report complete genome sequences of the BJ Group, including four isolates (Isolates BJ01, BJ02, BJ03, and BJ04) of the SARS-CoV.It is remarkable that all members of the BJ Group share a common haplotype, consisting of seven loci that differentiate the group from other isolates published to date. Among 42 substitutions uniquely identified from the BJ group, 32 are non-synonymous changes at the amino acid level. Rooted phylogenetic trees, proposed on the basis of haplotypes and other sequence variations of SARS-CoV isolates from Canada, USA, Singapore, and China, gave rise to different paradigms but positioned the BJ Group, together with the newly discovered GD01 (GD-Ins29) in the same clade, followed by the H-U Group (from Hong Kong to USA) and the H-T Group (from Hong Kong to Toronto), leaving the SP Group (Singapore) more distant. This result appears to suggest a possible transmission path from Guangdong to Beijing/Hong Kong, then to other countries and regions.

The M Protein of SARS-CoV: Basic Structural and Immunological Properties

We studied structural and immunological properties of the SARS-CoV M (membrane) protein, based on comparative analyses of sequence features, phylogenetic investigation, and experimental results. The M protein is predicted to contain a triple-spanning transmembrane (TM) region, a single N-glycosylation site near its N-terminus that is in the exterior of the virion, and a long C-terminal region in the interior. The M protein harbors a higher substitution rate (0.6% correlated to its size) among viral open reading frames (ORFs) from published data. The four substitutions detected in the M protein, which cause non-synonymous changes, can be classified into three types. One of them results in changes of pI (isoelectric point) and charge, affecting antigenicity. The second changes hydrophobicity of the TM region, and the third one relates to hydrophilicity of the interior structure. Phylogenetic tree building based on the variations of the M protein appears to support the non-human origin of SARS-CoV. To investigate its immunogenicity, we synthesized eight oligopeptides covering 69.2% of the entire ORF and screened them by using ELISA (enzyme-linked immunosorbent assay) with sera from SARS patients. The results confirmed our predictions on antigenic sites.

Genome Organization of the SARS-CoV

Annotation of the genome sequence of the SARS-CoV (severe acute respiratory syndrome-associated coronavirus) is indispensable to understand its evolution and pathogenesis. We have performed a full annotation of the SARS-CoV genome sequences by using annotation programs publicly available or developed by ourselves. Totally, 21 open reading frames (ORFs) of genes or putative uncharacterized proteins (PUPs) were predicted. Seven PUPs had not been reported previously, and two of them were predicted to contain transmembrane regions. Eight ORFs partially overlapped with or embedded into those of known genes, revealing that the SARS-CoV genome is a small and compact one with overlapped coding regions. The most striking discovery is that an ORF locates on the minus strand. We have also annotated non-coding regions and identified the transcription regulating sequences (TRS) in the intergenic regions. The analysis of TRS supports the minus strand extending transcription mechanism of coronavirus. The SNP analysis of different isolates reveals that mutations of the sequences do not affect the prediction results of ORFs.