Objective To prepare humanized monoclonal antibodies(Mabs)targeting Acinetobacter baumannii(Ab)based on single memory B cell sequencing technology,construct the immune repertoire of the core protein of Ab,hemolysin-coregulated protein(Hcp),and express its Mabs with binding activity.Methods E.coli BL21 harboring the recombinant plasmid pGEX-6p-1-Hcp was constructed.Hcp protein was obtained using protein expression and affinity chromatography.Female SPF BALB/c mice(6～8 weeks old,weighing 18～20 g)were immunized intramuscularly with antigen Hcp to generate specific memory B cells.Single antigen-specific memory B cells were sorted using flow cytometry.The immune repertoire of Hcp was constructed using single-cell sequencing technology,and bioinformatics analysis was performed on the sequencing results.Mabs were obtained using antibody humanization techniques.The in vitro binding activity of the antibodies was detected by ELISA.Results The target protein Hcp with a purity>95%was obtained after expression and purification.The immune repertoire of Hcp was successfully constructed,and the results of BCR clonotype identification and analysis,CDR3 region characteristic analysis,and V-J gene pairing characteristic analysis were achieved.Antibody humanization got 7 Mabs,that is,IgG1-1,IgG1-2,IgG2-1,IgG2-2,IgG3-1,IgG4-1 and IgG4-2.ELISA results showed IgG1-1,IgG3-1,IgG4-1,and IgG4-2 had an antibody binding titer of 1∶1 280,IgG2-2 of 1∶10 240,IgG2-1 of 1∶5 120,and IgG1-2 of 1∶160.Conclusion Single-cell sequencing technology enables rapid,accurate,and efficient construction of an Hcp protein immune repertoire containing extensive antibody information.Utilizing this immune repertoire allows for the expression of Mabs with binding activity.

Objective To explore the effect of Mycobacterium tuberculosis infection on the primary cilia of mono-cytse-macrophages and its potential mechanisms of promoting osteoclast differentiation.Methods Bone marrow-de-rived mononuclear cells(BMMCs)isolated from patients in control group and spinal tuberculosis group(TB group)were performed in vitro culture,and then cultured with Mycobacterium tuberculosis,infection model(Rv group)was constructed.Changes in cilia were observed by fluorescence staining and scanning electron microscopy tech-nique,a mouse spinal TB model was constructed for validating.Results Compared with the control group,the ex-pression of primary cili markers in the lesion of bone tissue of patients in TB group decreased significantly;After co-culturing with Mycobacterium tuberculosis,the ratio(48.56％±7.77％vs 9.58％±5.59％)and length(4.050[3.289,4.666]μm vs 0[0,0.676]μm)of primary cilia of monocytes-macrophages in the Rv group decreased sig-nificantly;The infiltration of osteoclasts in the bone marrow cavity of spinal TB mice was obvious,and the propor-tion and length of primary cilia decreased significantly.Conclusion Intracellular infection of Mycobacterium tuberculosis can induce degradation of primary cilia in monocytes-macrophages,promote osteoclast differentiation,and exacerbate vertebral bone resorption.

Objective To explore the effect of Mycobacterium tuberculosis infection on the primary cilia of mono-cytse-macrophages and its potential mechanisms of promoting osteoclast differentiation.Methods Bone marrow-de-rived mononuclear cells(BMMCs)isolated from patients in control group and spinal tuberculosis group(TB group)were performed in vitro culture,and then cultured with Mycobacterium tuberculosis,infection model(Rv group)was constructed.Changes in cilia were observed by fluorescence staining and scanning electron microscopy tech-nique,a mouse spinal TB model was constructed for validating.Results Compared with the control group,the ex-pression of primary cili markers in the lesion of bone tissue of patients in TB group decreased significantly;After co-culturing with Mycobacterium tuberculosis,the ratio(48.56％±7.77％vs 9.58％±5.59％)and length(4.050[3.289,4.666]μm vs 0[0,0.676]μm)of primary cilia of monocytes-macrophages in the Rv group decreased sig-nificantly;The infiltration of osteoclasts in the bone marrow cavity of spinal TB mice was obvious,and the propor-tion and length of primary cilia decreased significantly.Conclusion Intracellular infection of Mycobacterium tuberculosis can induce degradation of primary cilia in monocytes-macrophages,promote osteoclast differentiation,and exacerbate vertebral bone resorption.

Phage antibody display technology is currently the most widely used in vitro antibody screening technology,which uses bacteriophages as a vector,and inserts exogenous antibody library genes into phage capsid protein genes,and expresses the capsid protein on the phage surface while also displays the antibody protein.Antibody drugs play an important role in tumor immunity and microbial immunity due to their targeting advantages,which is also an important driving force for them to become a hot spot in the field of pharmaceutical research and development.Therefore,this article reviews the background,basic principles,antibody library types and antibody fragment types of phage display technology,and looks forward to the latest progress and application prospects of fully human antibodies.

2019 Novel coronavirus (2019-nCoV) infection caused a pandemic in the world. From the reported cases in the literatures, the level of D-dimer in patients with coronavirus disease 2019 (COVID-19) is positively correlated with the severity of illness, which needs the attention of clinical workers. According to Western medicine, the increase of D-dimer is related to the hyperactivity of fibrinolytic system and the shortening of prothrombin time (PT), resulting in excessive production and degradation of plasma fibrin and hypercoagulable state of blood, while traditional Chinese medicine (TCM) believes that the above syndromes belong to the pathogenesis of "blood stasis" according to TCM theories. Over the years, TCM has a significant effect on promoting blood circulation, removing blood stasis and improving microcirculation. This article reviews the mechanism, clinical significance, understanding of TCM and common methods of promoting blood circulation and removing blood stasis caused by 2019-nCoV, in order to provide ideas for the prevention and treatment of impaired blood coagulation in patients with COVID-19.

Support vector machine (SVM) has shown its excellent learning and generalization ability for the binary classification of real problems and has been extensively employed in many areas. In this paper, SVM, K-Nearest Neighbor, Decision Tree C4.5 and Artificial Neural Network were applied to identify cancer patients and normal individuals using the concentrations of 6 elements including macroelements (Ca, Mg) and microelements (Ba, Cu, Se, Zn) in human blood. It was demonstrated, by using the normalized features instead of the original features, the classification performances can be improved from 91.89% to 95.95%, from 83.78% to 93.24%, and from 90.54% to 94.59% for SVM, K-NN and ANN respectively, whereas that of C4.5 keeps unchangeable. The best average accuracy of SVM with linear dot kernel by using 5-fold cross validation reaches 95.95%, and is superior to those of other classifiers based on K-NN (93.24%), C4.5 (79.73%), and ANN (94.59%). The study suggests that support vector machine is capable of being used as a potential application methodology for SVM-aided clinical cancer diagnosis.
Algorithms ; Barium ; blood ; Calcium ; blood ; Computational Biology ; methods ; Copper ; blood ; Diagnosis, Computer-Assisted ; methods ; Humans ; Neoplasms ; blood ; diagnosis ; Neural Networks (Computer) ; Trace Elements ; blood

BACKGROUND: Auditory steady-state responses (ASSR) is an objective method of hearing examination in clinic in recent years. ASSR has the frequency specificity as compared with previous auditory brainstem responses (ABR).OBJECTIVE: To investigate the accuracy of ASSR in objective hearing assessment.DESIGN: A case-control observation.SETTING: Department of Otorhinolaryngology, the First Affiliated Hospital of Sun Yat-sen University.PARTICIPANTS: The subjects in the normal hearing group were the 21 undergraduates (42 ears) were enrolled, they all had not any symptoms of ear disease, without history of noise exposure and disease of vestibule system, and they were normal in otoscopy. The outpatients and inpatients with neurosensory deafness were selected from the Department of Otorhinolaryngology, the First Affiliated Hospital of Sun Yat-sen University. All the children cases worn hearing aids, and had the speech ability, and cooperated in the examination. The main types included 6 ears of sudden deafness,8 ears of presbycusis, and 20 ears of neurosensory deafness due to other unknown causes. Central lesions were excluded by MR examination, and all the patients agreed with the enrollment. The results of pure-tone audiometry were all flat or descending audiogram. According to the severity of hearing damage, the patients were divided into mild deafness group (13ears), moderate deafness group (9 ears) and moderate-to-severe deafness group (12 ears).METHODS: ① The pure-tone audiometry was performed at the frequencies of 0.125-8 000 Hz in a sound insulation room. The auditory threshold grades of the subjects with normal hearing all accorded with the standards of GB-7583-87 expected value distribution. The average value of air-conduction auditory thresholds of pure-tone audiometry at the frequencies of 0.5, 1, 2 and 4 kHz was calculated. ② ASSR measurement was performed with the synchronous stimulation pattern in a sound and electromagnetic shielding room, including 8 points for both ears of the same stimulation intensity and the carrier frequency tones of 0.5, 1, 2 and 4 kHz respectively.③ ABR examination was performed by click sounds with sparse waves in a sound and electromagnetic shielding room, and insert earphones were used.The threshold results were judged according to the minimal stimulation sound intensity of the distinguishable Ⅴ wave. ③ The results of pure-tone audiometry were compared with those of ABR examination, and the results of ASSR measurement in different hearing groups were processed with analysis of variance, multi-classification discrimination based Bayes standard and q test.MAIN OUTCOME MEASURES: The thresholds of pure-tone audiometry, ASSR measurement and ABR examination, and the correct rate analyzed by the multi-classification discrimination based Bayes standard were mainly observed.RESULTS: The indexes of the 42 ears in the normal hearing group, 13, 9 and 12 ears in the mild, moderate and moderate-to-severe deafness groups were all involved in the analysis of results. ① The ABR values were accorded with the actual hearing levels, and the closest to the ASSR thresholds at 1-2 kHz; ASSR reflected induction rates at different frequencies were gradually decreased with the aggravation of hearing damage, and that at each frequency varied with the changes of hearing level, the induction rates of ASSR responses were all 100% for the subjects with normal hearing and patients with mild deafness, but those for the patients with moderate and moderate-to-severe deafness were decreased (0.5 kHz: 77.8%,92.8%; 4 kHz: 88.9%, 85.7%). At different frequencies, the ASSR thresholds in the moderate-to-severe deafness group were significantly higher than those in the normal hearing group (P ＜ 0.05). The ASSR thresholds at 0.5 and 4 kHz in the moderate-to-severe deafness group were significantly higher than those in the mild deafness group (P ＜ 0.05). The ASSR threshold at 2 kHz in the mild deafness group was significantly higher than that in the normal hearing group (P ＜ 0.05). The ASSR thresholds at 4 kHz in the everedeafness group were significantly higher than those in the normal hearing group and mild deafness group. ② The incorrect discriminations of actual pure-tone audiometry were analyzed with the interactive clustering discriminant analysis of ASSR measurement and actual pure-tone audiometry, and the results showed that the correct rate of discrimination was 100% in the normal hearing group; Only 1 of the 12 cases in the mild deafness group was incorrectly judged, and the correct rate was 92%; Only 1 of the 19 cases in the moderate deafness group was incorrectly judged, and the correct rate was 89%; the correct rate in the moderateto-severe deafness group was 83%.CONCLUSION: The results of ASSR measurement can detect the incorrect discrimination of objective hearing condition by taking the results of pure-tone audiometry as the standards. ASSR has an acceptable accuracy for deafness higher than mild level in estimating objective hearing, and it has a better prospect of application in practice.