Transcriptome sequencing of the endangered land snail Karaftohelix adamsi from the Island Ulleung: De novo assembly, annotation, valuation of fitness genes and SSR markers.

BACKGROUND: The Bradybaenidae snail Karaftohelix adamsi is endemic to Korea, with the species tracked from Island Ulleung in North Gyeongsang Province of South Korea. K. adamsi has been classified under the Endangered Wildlife Class II species of Korea and poses a severe risk of extinction following habitat disturbances. With no available information at the DNA (genome) or mRNA (transcriptome) level for the species, conservation by utilizing informed molecular resources seems difficult. OBJECTIVE: In this study, we used the Illumina short-read sequencing and Trinity de novo assembly to draft the reference transcriptome of K. adamsi. RESULTS: After assembly, 13,753 unigenes were obtained of which 10,511 were annotated to public databases (a maximum of 10,165 unigenes found homologs in PANM DB). A total of 6,351, 3,535, 358, and 3,407 unigenes were ascribed to the functional categories under KOG, GO, KEGG, and IPS, respectively. The transcripts such as the HSP 70, aquaporin, TLR, and MAPK, among others, were screened as putative functional resources for adaptation. DNA transposons were found to be thickly populated in comparison to retrotransposons in the assembled unigenes. Further, 2,164 SSRs were screened with the promiscuous presence of dinucleotide repeats such as AC/GT and AG/CT. CONCLUSION: The transcriptome-guided discovery of molecular resources in K. adamsi will not only serve as a basis for functional genomics studies but also provide sustainable tools to be utilized for the protection of the species in the wild. Moreover, the development of polymorphic SSRs is valuable for the identification of species from newer habitats and cross-species genotyping.

Transcriptome analysis of Haemaphysalis flava female using Illumina HiSeq 4000 sequencing: de novo assembly, functional annotation and discovery of SSR markers.

BACKGROUND: Ticks are ectoparasites capable of directly damaging their hosts and transmitting vector-borne diseases. The ixodid tick Haemaphysalis flava has a broad distribution that extends from East to South Asia. This tick is a reservoir of severe fever with thrombocytopenia syndrome virus (SFTSV) that causes severe hemorrhagic disease, with cases reported from China, Japan and South Korea. Recently, the distribution of H. flava in South Korea was found to overlap with the occurrence of SFTSV. METHODS: This study was undertaken to discover the molecular resources of H. flava female ticks using the Illumina HiSeq 4000 system, the Trinity de novo sequence assembler and annotation against public databases. The locally curated Protostome database (PANM-DB) was used to screen the putative adaptation-related transcripts classified to gene families, such as angiotensin-converting enzyme, aquaporin, adenylate cyclase, AMP-activated protein kinase, glutamate receptors, heat shock proteins, molecular chaperones, insulin receptor, mitogen-activated protein kinase and solute carrier family proteins. Also, the repeats and simple sequence repeats (SSRs) were screened from the unigenes using RepeatMasker (v4.0.6) and MISA (v1.0) software tools, followed by the designing of SSRs flanking primers using BatchPrimer 3 (v1.0) software. RESULTS: The transcriptome produced a total of 69,822 unigenes, of which 46,175 annotated to the homologous proteins in the PANM-DB. The unigenes were also mapped to the EuKaryotic Orthologous Groups (KOG), Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) specializations. Promiscuous presence of protein kinase, zinc finger (C2H2-type), reverse transcriptase, and RNA recognition motif domains was observed in the unigenes. A total of 3480 SSRs were screened, of which 1907 and 1274 were found as tri- and dinucleotide repeats, respectively. A list of primer sequences flanking the SSR motifs was detailed for validation of polymorphism in H. flava and the related tick species. CONCLUSIONS: The reference transcriptome information on H. flava female ticks will be useful for an enriched understanding of tick biology, its competency to act as a vector and the study of species diversity related to disease transmission.

A review of the endangered mollusks transcriptome under the threatened species initiative of Korea.

Transcriptome studies for conservation of endangered mollusks is a proactive approach towards managing threats and uncertainties facing these species in natural environments. The population of these species is declining due to habitat destruction, illicit wildlife trade, and global climate change. These activities risk the free movement of species across the wild landscape, loss of breeding grounds, and restrictions in displaying the physiological attributes so crucial for faunal welfare. Gastropods face the most negative ecological effects and have been enlisted under Korea's protective species consortium based on their population dynamics in the last few years. Moreover, with the genetic resources restricted for such species, conservation by informed planning is not possible. This review provides insights into the activities under the threatened species initiative of Korea with special reference to the transcriptome assemblies of endangered mollusks. The gastropods such as Ellobium chinense, Aegista chejuensis, Aegista quelpartensis, Incilaria fruhstorferi, Koreanohadra kurodana, Satsuma myomphala, and Clithon retropictus have been represented. Moreover, the transcriptome summary of bivalve Cristaria plicata and Caenogastropoda Charonia lampas sauliae is also discussed. Sequencing, de novo assembly, and annotation identified transcripts or homologs for the species and, based on an understanding of the biochemical and molecular pathways, were ascribed to predictive gene function. Mining for simple sequence repeats from the transcriptome have successfully assisted genetic polymorphism studies. A comparison of the transcriptome scheme of Korean endangered mollusks with the genomic resources of other endangered mollusks have been discussed with homologies and analogies for dictating future research.

Creep mechanical tests and shear rheological model of the anchorage rock mass under water‒rock coupling.

The development of deep geotechnical engineering is restricted by the complex geological conditions of deep rock masses and the unknown creep mechanism of rock in water-rich environments. To study the shear creep deformation law of the anchoring rock mass under different water content conditions, marble was used as the bedrock to make anchoring specimens, and shear creep tests of the anchoring rock mass under different water contents were carried out. The influence of water content on rock rheological characteristics is explored by analysing the related mechanical properties of the anchorage rock mass. The coupling model of the anchorage rock mass can be obtained by connecting the nonlinear rheological element and the coupling model of the anchorage rock mass in series. Related studies show that (1) shear creep curves of anchorage rock masses under different water contents have typical creep characteristics, including decay, stability and acceleration stages. The creep deformation of the specimens can be improved with increasing moisture content. (2) The long-term strength of the anchorage rock mass shows an opposite change law with increasing water content. The creep rate of the curve increases gradually with increasing water content. The creep rate curve shows a U-shaped change under high stress. (3) The nonlinear rheological element can explain the creep deformation law of rock in the acceleration stage. By connecting the nonlinear rheological element with the coupled model of anchoring rock mass in series, the coupled model of water‒rock under water cut conditions can be obtained. The model can be used to study and analyse the whole process of shear creep of an anchored rock mass under different water contents. This study can provide theoretical support for the stability analysis of anchor support tunnel engineering under water cut conditions.

Transcriptome analysis of the endangered dung beetle Copris tripartitus (Coleoptera: Scarabaeidae) and characterization of genes associated to immunity, growth, and reproduction.

BACKGROUND: Dung beetles recycle organic matter through the decomposition of feces and support ecological balance. However, these insects are threatened by the indiscriminate use of agrochemicals and habitat destruction. Copris tripartitus Waterhouse (Coleoptera: Scarabaeidae), a dung beetle, is listed as a class-II Korean endangered species. Although the genetic diversity of C. tripartitus populations has been investigated through analysis of mitochondrial genes, genomic resources for this species remain limited. In this study, we analyzed the transcriptome of C. tripartitus to elucidate functions related to growth, immunity and reproduction for the purpose of informed conservation planning. RESULTS: The transcriptome of C. tripartitus was generated using next-generation Illumina sequencing and assembled de novo using a Trinity-based platform. In total, 98.59% of the raw sequence reads were processed as clean reads. These reads were assembled into 151,177 contigs, 101,352 transcripts, and 25,106 unigenes. A total of 23,450 unigenes (93.40%) were annotated to at least one database. The largest proportion of unigenes (92.76%) were annotated to the locally curated PANM-DB. A maximum of 5,512 unigenes had homologous sequences in Tribolium castaneum. Gene Ontology (GO) analysis revealed a maximum of 5,174 unigenes in the Molecular function category. Further, in Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis, a total of 462 enzymes were associated with established biological pathways. Based on sequence homology to known proteins in PANM-DB, representative immunity, growth, and reproduction-related genes were screened. Potential immunity-related genes were categorized into pattern recognition receptors (PRRs), the Toll-like receptor signaling pathway, the MyD88- dependent pathway, endogenous ligands, immune effectors, antimicrobial peptides, apoptosis, and adaptation-related transcripts. Among PRRs, we conducted detailed in silico characterization of TLR-2, CTL, and PGRP_SC2-like. Repetitive elements such as long terminal repeats, short interspersed nuclear elements, long interspersed nuclear elements and DNA elements were enriched in the unigene sequences. A total of 1,493 SSRs were identified among all unigenes of C. tripartitus. CONCLUSIONS: This study provides a comprehensive resource for analysis of the genomic topography of the beetle C. tripartitus. The data presented here clarify the fitness phenotypes of this species in the wild and provide insight to support informed conservation planning.

Prediction of microvascular invasion in hepatocellular carcinoma with magnetic resonance imaging using models combining deep attention mechanism with clinical features / 南方医科大学学报

OBJECTIVE@#To investigate the consistency and diagnostic performance of magnetic resonance imaging (MRI) for detecting microvascular invasion (MVI) of hepatocellular carcinoma (HCC) and the validity of deep learning attention mechanisms and clinical features for MVI grade prediction.@*METHODS@#This retrospective study was conducted among 158 patients with HCC treated in Shunde Hospital Affiliated to Southern Medical University between January, 2017 and February, 2020. The imaging data and clinical data of the patients were collected to establish single sequence deep learning models and fusion models based on the EfficientNetB0 and attention modules. The imaging data included conventional MRI sequences (T1WI, T2WI, and DWI), enhanced MRI sequences (AP, PP, EP, and HBP) and synthesized MRI sequences (T1mapping-pre and T1mapping-20 min), and the high-risk areas of MVI were visualized using deep learning visualization techniques.@*RESULTS@#The fusion model based on T1mapping-20min sequence and clinical features outperformed other fusion models with an accuracy of 0.8376, a sensitivity of 0.8378, a specificity of 0.8702, and an AUC of 0.8501 for detecting MVI. The deep fusion models were also capable of displaying the high-risk areas of MVI.@*CONCLUSION@#The fusion models based on multiple MRI sequences can effectively detect MVI in patients with HCC, demonstrating the validity of deep learning algorithm that combines attention mechanism and clinical features for MVI grade prediction.

The relationship of α-hydroxybutyrate dehydrogenase with 1-year outcomes in patients with intracerebral hemorrhage: A retrospective study.

Background and aims: Cardiac enzymes are recognized as a valuable tool for predicting the prognosis of various cardiovascular diseases. The prognostic value of alpha-hydroxybutyrate dehydrogenase (α-HBDH) in patients with intracerebral hemorrhage (ICH) was ambiguous and not evaluated. Methods: Two hundred and thirteen Chinese patients with ICH participated in the study from December 2018 to December 2019. Laboratory routine tests and cardiac enzymes, including α-HBDH level, were examined and analyzed. All the patients were classified into two groups by the median value of α-HBDH: B1 <175.90 and B2 ≥175.90 U/L. The clinical outcomes included functional outcome (according to modified Rankin Scale (mRS) score ≥3), all-cause death, and recurrent cerebro-cardiovascular events 1 year after discharge. Associations between the α-HBDH and the outcomes were evaluated using logistic regression analysis. Univariate survival analysis was performed by the Kaplan-Meier method and log-rank test. Results: Of the 213 patients, 117 had α-HBDH ≥175.90 U/L. Eighty-two patients had poor functional outcomes (mRS≥3). During the 1-year follow-up, a total of 20 patients died, and 15 of them had α-HBDH ≥175.90 U/L during the follow-up time. Moreover, 24 recurrent events were recorded. After adjusting confounding factors, α-HBDH (≥175.90) remained an indicator of poor outcome (mRS 3-6), all-cause death, and recurrent cerebro-cardiovascular events. The ORs for B2 vs. B1 were 4.78 (95% CI: 2.60 to 8.78, P = 0.001), 2.63 (95% CI: 0.80 to 8.59, P = 0.11), and 2.40 (95% CI: 0.82 to 7.02, P = 0.11) for poor functional outcomes with mRS ≥ 3, all-cause death, and recurrent cerebro-cardiovascular events, respectively. Conclusion: Increased α-HBDH at admission was independently related to poor functional outcome and all-cause mortality in patients with ICH at 1-year follow-up.

Predictive value of the apolipoprotein B/A1 ratio in intracerebral hemorrhage outcomes.

BACKGROUND AND AIMS: The apolipoprotein B (apoB)/apolipoprotein A1 (apoA1) ratio is a key indicator in predicting future cardiovascular outcomes. However, it is still unclear whether the ratio of apoB/apoA1 is a better predictor of the outcomes after intracerebral hemorrhage (ICH). Therefore, we aimed to assess the relationships between the ratio of apoB/apoA1 and functional outcomes, all-cause mortality, and stroke recurrence in ICH patients. METHODS: Two hundred and sixteen Chinese ICH patients participated in this study from December 2018 to December 2019. Laboratory routine tests including hematology analysis, coagulation tests, and lipid levels were examined. The clinical outcomes included functional outcomes evaluated by the modified Rankin Scale score (mRS), all-cause death, and stroke recurrence 1 year after discharge. Associations between the apoB/apoA1 ratio and the outcomes were evaluated using logistic regression analysis. Based on multivariate analysis, we constructed a nomogram. Univariate survival analysis was performed by the Kaplan-Meier method and log-rank test. All the patients were classified into two groups by the median value of the apoB/apoA1 ratio: B1 < 0.8 and B2 ≥ 0.8. RESULTS: Of the 216 patients, 107 had an apoB/apoA1 ratio ≥ 0.8. Eighty-five patients had poor functional outcomes (mRS ≥ 3), and 32 patients had severe functional outcomes (mRS ≥ 4). During the 1-year follow-up, a total of 18 patients died, and 13 patients had apoB/apoA1 ratio levels ≥0.8 during the 1-year follow-up period. Moreover, 16 recurrent strokes were recorded. Adjustments for age, sex, smoking, alcohol, body mass index, lipid levels, and hematoma site and volume showed that a high apoB/apoA1 ratio was significantly related to adverse functional outcomes and all-cause mortality. The ORs for B2 versus B1 were 3.76 (95% CI: 1.37 to 10.40, p = 0.010), 22.74 (95% CI: 1.08 to 474.65, p = 0.044), and 7.23 (95% CI: 1.28 to 40.88, p = 0.025) for poor functional outcomes with mRS ≥ 3, mRS ≥ 4, and all-cause mortality, respectively. CONCLUSION: An increased apoB/apoA1 ratio at admission was independently related to poor functional outcome and all-cause mortality in ICH patients at the 1-year follow-up.

Clinical characteristics and risk factors of ischemic stroke in young adults / 中国综合临床

Objective:To analyze the clinical characteristics and risk factors of ischemic stroke in young adults.Methods:A retrospective analysis was conducted on 80 ischemic stroke patients (age ≤45 years) admitted to Beijing Tiantan Hospital from March 2019 to October 2019 as the young stroke group, and 117 ischemic stroke patients (age >45 years) hospitalized during the same period as the middle-aged and elderly stroke group. The blood test indexes of the two groups were compared, and the risk factors related to stroke, including smoking history, drinking history, hypertension, hyperlipidemia and diabetes history, were compared and analyzed. Two sets of independent sample t-test, Mann-Whitney U-test or χ2 test were used to compare the above indicators of patients in the two groups. Results:The activated partial prothrombin time, protein S, uric acid, homocysteine and D-dimer levels in middle-aged and elderly stroke group were (29.73±3.40) s, (105.58±27.23) %, (297.29±85.99) μmol/L, (17.58±14.45) μmol/L and (2.75±3.08) mg/L, respectively. Compared with the middle-aged and elderly stroke group, the young stroke group had higher activated partial thrombin time (31.51±6.75) s, protein S (115.20±26.97) %, uric acid (326.82±93.51) μmol/L, homocysteine (22.63±16.98) μmol/L and lower D dimer level of (1.19±2.88) mg/L compared with the elder group, the difference between the two groups was statistically significant ( t values were 2.17, 2.01, 2.20, 2.14 and 2.13, respectively, P values were 0.032, 0.046, 0.029, 0.039 and 0.034, respectively). The positive rate of lupus anticoagulant in young stroke group was 12.5% (4/32), which was higher than 1.8% (1/57) in middle-aged and elderly stroke group, and there was significant difference between the two groups (χ 2=4.46, P=0.035). The proportions of smoking and drinking in young stroke group were 63.8% (51/80) and 62.5% (50/80), respectively, which were higher than 49.6% (58/117) and 47.9% (56/117) in middle-aged and elderly stroke group, and there was significant difference between the two groups (χ 2 values were 3.86 and 4.09; P values were 0.04 and 0.04). The proportion of hypertension and diabetes in young stroke group was 48.8% (39/80) and 17.5%(14/80), respectively, which were lower than 63.2%(74/117) and 30.8%(36/117) in middle-aged and elderly stroke group, and there was significant difference between the two groups (χ 2 values were 4.08 and 4.56; P values were 0.043 and 0.033). According to the levels of uric acid and homocysteine, young stroke was divided into different subgroups and compared.The creatinine level of high uric acid group (≥416 μmol/L) was (90.08±28.46) mmol/L, which was higher than that of normal uric acid group (<416 μmol/L) of (63.37±22.2) mmol/L. There was significant difference between the two groups ( t value was 2.23, P value was 0.046). The levels of fibrinogen and creatinine in high homocysteine group (≥15 μmol/L) were (3.27±1.09) g/L and (72.13±28.69) mmol/L, respectively which were significantly higher than those in normal homocysteine group (<15 μmol/L) of (2.78±0.67) g/L and (58.92±12.08) mmol/L, There was significant difference between the two groups (the t values were 2.32 and 2.51; P values were 0.023 and 0.014). Conclusions:Compared with middle-aged and elderly stroke, young ischemic stroke has higher levels of prothrombin time, protein S, uric acid and homocysteine, lower levels of D dimer and higher positive rate of lupus anticoagulant. At the same time, the proportion of smoking and drinking was higher in young stroke group, but the proportion of hypertension and diabetes was relatively lower.

Clinical study of ultrasound evaluation of fiberoptic bronchoscope guided tracheotomy / 中华危重病急救医学

Objective:To explore the clinical effect of ultrasound evaluation of fiberoptic bronchoscope (FB) guided tracheotomy, which can provide help for difficult tracheotomy and new operators.Methods:The operating protocol was standardized for ultrasound evaluation of FB guided tracheotomy. Ten patients with difficult tracheotomy admitted to the department of critical care medicine of Donge Hospital from October 2019 to January 2020 were enrolled. According to this protocol, FB guided tracheotomy was performed under the ultrasound evaluation, and the amount of blood loss, operation time and related complications during procedures were collected.Results:The preparation of supplies, personnel, patients and the operation, the process of FB guided tracheotomy under the ultrasound evaluation were standardized. When tracheotomy was preformed for patients with difficult tracheotomy, it was necessary to use ultrasound first to evaluate the neck condition and vascular disorientation of patients, and the tracheotomy plan (tracheotomy site, incision size, and incision depth) was designed, and then the tracheotomy process was monitored under the guidance of FB. Among the 10 patients with difficult tracheotomy, 6 were male and 4 were female; body mass index was (32.2±1.4) kg/m 2. Tracheotomy was successfully completed within 10 minutes in all the 10 patients, with less than 5 mL blood loss, and no complications occurred. Conclusion:Ultrasound evaluation of FB guided tracheotomy can improve the clinical operations and ensure patient safety.

EV71 induces incomplete cellular autophagy to promote self-replication / 中华实验和临床病毒学杂志

Objective@#To investigate the relationship between enterovirus 71 (EV71) and autophagy and the effect of autophagy on viral replication.@*Methods@#RD cells were pretreated with rapamycin, Chloroquine (CQ) and 3-methyladenine (3-MA). The effects of different stages of autophagy on viral replication were detected by Western blotting and plaque assay.@*Results@#The study showed that EV71 infection could induce incomplete autophagy in RD cells. Replication of EV71 was promoted in the CQ treatment group, while the level of replication of the rapamycin and 3-MA treatment groups was reduced. Further studies have shown that mTOR is a key molecule affecting EV71 replication, and inhibition of mTOR by rapamycin can inhibit the synthesis of viral RNA.@*Conclusions@#The effects of different stages of autophagy on viral replication are also different. Inhibition of autophagic lysosome degradation promotes viral replication, while inhibition of the early stage of autophagy or promotion of autophagy reduces the level of viral replication. mTOR can affect the replication of EV71 at RNA level.

Sequence analysis of enterovirus 71 3′UTR and construction of a recombinant cDNA infectious clone / 中华实验和临床病毒学杂志

Objective@#To sequence the 3′UTR of enterovirus 71 strains, investigate its foundation and impact in virulence by constructing a 3′UTR-replaced recombinant cDNA infectious clone.@*Methods@#Viral RNA of EV-A71 isolated viruses were extracted, and the nucleotide analysis was performed after sequencing. The 3′UTR of a full-length infectious clone of SDLY107 strain was replaced by its corresponding part of SDLY1 strain, and then the recombinant virus was constructed and identified.@*Results@#The nine isolated strains were classified into sub-genotype C4a of enterovirus (EV)-A71 by analysis, and nucleotide sequence homology for 3′UTR were 94%-100%. 3′UTR of EV-A71 strains may be associated with its pathogenicity. Identification of the rescued virus by sequencing and indirect immunofluorescence confirmed the successful construction of infectious recombinant virus.@*Conclusions@#Sequence analysis indicated that the 3′UTR may be involved in the pathogenicity of EV-A71. The recombinant virus SDLY107(1-3′UTR) was rescued successfully. Our study may provide evidence for further research on the influence of 3′UTR on the virulence of enterovirus 71.

The 461th case: fever, hematuria, and right lumbar pain / 中华内科杂志

A 56-year-old female was admitted to the Department of Rheumatology,Peking Union Medical College Hospital with complaint of recurrent fever and acute lumbar pain.Fever was complicated with malaise,cough and occasional blood-streaked sputum.Lab tests showed elevated white blood cell count,increased serum creatinine,erythrocyte sedimentation rate and C-reactive protein.Other lab findings included severe anemia,hematuria,and proteinuria.Immunological examinations were positive for antinuclear antibodies,antineutrophil cytoplasmic antibodies and antiglomerular basement membrane antibody.Ultrasonography and CT scan detected a huge spontaneous perirenal hematoma at right side.Angiography revealed multiple microaneurysms on bilateral renal arteries and branches.A diagnosis of systemic vasculitis was suggested.Under the combination therapy of corticosteroids and cyclophosphamide,the patient presented sustained remission for one year.This case indicates that prompt and sufficient treatment of primary disease is essential to a promising outcome.

Correlation of insulin-like growth factor 1 expression in placenta with DNA methylation and fetal macrosomia / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To explore the correlation between methylation of insulin-like growth factor 1 (IGF-1) gene promoter and its placenta-specific expression and fetal macrosoma.</p><p><b>METHODS</b>One hundred twenty nine healthy pregnant women were recruited between April 2011 and March 2012. Baseline data were collected with self-report questionnaires. Real-time quantitative PCR was used to determine the expression of IGF-1 mRNA in the placenta. Methylation level of the IGF 1 gene was determined with matrix-assisted laser desorption/ionization-time of flight mass spectrometry.</p><p><b>RESULTS</b>The expression of IGF-1 in placenta and its methylation level showed no significant difference between macrosomic fetuses and controls. No linear correlation was found between IGF-1 mRNA expression and methylation level of IGF-1 promoter (r=0.128, P=0.295). IGF-1 promoter region in placenta showed a hypomethylation status. However, a positive correlation was found between IGF-1 expression and birth weight below 4260 g (r=0.264, P=0.022). The expression of IGF-1 mRNA was significantly higher in those with a birth weight below 4260 g, which suggested that placental IGF-1 expression may contribute to increased birth weight. In regard to fetal overgrowth, however, there seemed to be a negative correlation in which placental IGF-1 expression was downregulated to limit fetal overgrowth.</p><p><b>CONCLUSION</b>No linear correlation was found between placental IGF-1 expression and methylation level of IGF-1 promoter with a hypomethylation status. The contribution of placental IGF-1 expression to birth weight is bidirectional. Increased expression seems to promote fetal growth, while decreased expressions may curb overgrowth, therefore control fetal growth in a relatively normal range.</p>

Mechanism and process optimization of ultrafiltration of residual DNA in rabies vaccine / 军事医学

Objective To control residual DNA by optimizing methodology during the production of rabies vaccine using Vero cells as a vector .Methods The antigen recovery rate was assessed by linked immunosorbent assay-sandwich technique while the residual DNA was detected by DNA probe hybridization method .Antigen recovery and removal of DNA were the main indexes for evaluateing ultrafiltration , the vital part of rabies vaccine production .Three key factors in ultrafiltration were assessed: selection of membrane packages , ultrafiltration pressure and the concentration ratio .Then protamine was used to pretreat ultrafiltrates .Based on the two indicators mentioned above , the effect of protamine pretreat-ment on the ultrafiltrate was evaluated .Results and Conclusion The optimum condition of ultrafiltration was obtained on the basis of the general antigen recovery rate , DNA removal rate and actual production .The primary parameters of ultrafil-tration were as follows:7.5 ×105 ultrafiltration membrane packages, 20 times concentrated, 15 psi ultrafiltration pressure. After pretreatment with protamine , ultrafiltration has proved to be a molecular sieve in intercepting DNA ,while protamine can tangle the fragmented DNA and form a larger molecular segment , which is believed to be more conducive to ultrafiltra-tion interception .