Analysis of microbial diversity in the midgut of Haemaphysalis longicornis infected with severe fever with thrombocytopenia syndrome virus / 中国热带医学

@#Abstract: Objective　To investigate the composition and diversity of midgut microbial community of Haemaphysalis longicornis infected with severe fever with thrombocytopenia syndrome virus (SFTSV)． Methods　The midgut DNA of three group Haemaphysalis longicornis infected with SFTSV was extracted, and the 16S rDNA gene of the sample was sequenced by HiSeq platform. The composition and diversity of endosymbiotic microbial community were clarified by OTU cluster analysis and alpha diversity analysis. Results　The midgut microbial clusters of the three groups infected with SFTSV were 143, 113, 163 OTUs respectively; the sparsity curve and abundance grade curve showed that the data had sufficient sequencing depth, and the midgut of Haemaphysalis longicornis infected with SFTSV was rich in microbial composition, but the species distribution was uneven. The analysis of microbial community composition showed that Proteobacteria, Firmicutes and Actinobacteria were the main dominant bacteria at the phyla level. At the class level, Gammaproteobacteria, Bacilli, Betaproteobacteria and Actinomycetia were the main dominant bacteria. At the order level, Legionellales, Bacillales, Burkholderiales and Actinomycetales were the main dominant orders. At the family level, Coxiellaceae, Bacillaceae, Moraxellaceae and Rhodococcaceae were the main dominant families. At the genus level, the relative abundance of Coxiella was the highest, followed by Aeribaillus and Azonexus. Alpha diversity analysis showed that the average Shannon index was 139.67, the average Simpson index was 0.48, the average Chao index was 145.06, and the average ACE index was 147.11. Conclusions　The species diversity of intestinal microorganisms in Haemaphysalis longicornis infected with SFTSV is rich. The results provide a basis for further exploring the interaction between intestinal microbes of Haemaphysalis longicornis and SFTSV and developing new ideas for the prevention and control of ticks and tick-borne diseases.

In vitro transcribed (IVT) mRNA drugs: technical progress and application prospect / 药学学报

The in vitro transcribed (IVT) mRNA technology has progressed rapidly and the application of mRNA vaccines in the COVID-19 pandemic made it become the most talked-about topic. Compared with protein drugs, IVT mRNA has a lower cost; it can be modular produced and its sequence can be modified easily, so it has a broad application prospect. However, due to its short history, mRNA drugs face the problem of lacking sufficient clinical data, and there is no quality control standard for mRNA drugs except mRNA vaccines. We overview the sequence design, delivery vectors, administration, application prospect and safety considerations of mRNA drugs. We also discussed the quality control of mRNA drugs briefly.

A method for measuring the ADCP potency of anti-CD38 monoclonal antibody / 药学学报

A method to measure the antibody-dependent cell-mediated phagocytosis (ADCP) potency of anti-CD38 mAb was developed based on design of experiment (DoE) with a Jurkat/NFAT/CD32a-FcεRIγ transgenic cell line as the effector cell, the Daudi cell line as the target cells, and luciferase as the detection system. The DoE method was used for optimization of experimental parameters and methodological validation. The results show that anti-CD38 mAb exhibits a dose-response relationship with the following four-parameter equation: y = (A - D) / [1 + (x / C)B] + D. Several experimental parameters were optimized by statistical experimental design and determined as follows: the working concentration of anti-CD38 mAb was 800-20.81 ng·mL-1, the density of the target cells was 7.5×104 per well, and the density of effector cells was 2.5×104 per well, with an induction time of 6 h. The method showed good specificity. The recovery rate for samples from 5 different groups showed that the relative potencies of anti-CD38 mAb were (59.97 ± 4.74) %, (82.44 ± 5.15) %, (110.69 ± 11.71) %, (129.23 ± 5.22)% and (162.15 ± 3.66) %. The recoveries ranged from 103% to 120% and the RSDs of the above results were all less than 11%. The linear detection range was 50%-150%. Based on DoE design, this method for measuring ADCP potency of anti-CD38 mAb was optimized and validated with good specificity, repeatability and accuracy. This method can be used for evaluation of ADCP biological activity of anti-CD38 mAbs.

Value assignment study on bioactivity of monoclonal antibody working reference standard / 药学学报

The bioactivity of a working reference standard was determined by replicate bioassays with calibration against a primary reference standard. In this study the number of bioassay replicates needed for calibration first was calculated theoretically, and if the mean value of the experimental bioassay replicates fell within the predefined bioactivity level the bioactivity of the working reference was defined as 100%. Our results showed that when the total intermediate precision of the bioassay method was at 11.66% and the predefined bioactivity level was set at 95%-105% with a confidence level of 95%, 21 bioassay replicates should be carried out for calibration. The average value of the 22 experimental bioassay replicates was 101.96%, so the bioactivity of the working reference standard was consistent with that of the primary reference standard at 100%. The results suggest that a strategy of first calculating the number of bioassay replicates needed for calibration and then determining whether the resulting experimental mean value is within the predefined bioactivity level will be of value to the biopharmaceutical industry.

Interlaboratory method validation of HPLC-FMA for determination of polysorbate 80 in monoclonal antibodies / 药学学报

The high performance liquid chromatography-fluorescence micelle assay (HPLC-FMA) method for the content determination of polysorbate 80 in monoclonal antibody drugs was validated to study its applicability and transferability between various laboratories, and the feasibility to be included in the Chinese Pharmacopoeia. Both J.T. Baker and Nanjing Well-sourced polysorbate 80 was used in the collaborative validation of polysorbate 80 content analysis in seven different laboratories. The results show that when the protein concentration was no more than 20 mg·mL-1 and the concentration of polysorbate 80 ranged from 0.05 to 0.5 mg·mL-1, the method had good specificity. The recovery rates of the spiked samples ranged from 92.20% to 117.70% for J.T.Baker and from 93.90% to 117.20% for Nanjing Well. The intra-laboratory precision (%RSD) was less than 4.30% for J.T. Baker, and less than 2.60% for Nanjing Well, while the overall precision was less than 5.45% for J.T. Baker, and less than 6.70% for Nanjing Well. The linear correlation coefficient was more than 0.98 for J.T. Baker and more than 0.99 for Nanjing Well. The results of the collaborative validation prove that the HPLC-FMA method has good accuracy, precision, linearity, and specificity, and could be used for release control analysis of polysorbate 80 content in monoclonal antibodies across different laboratories.

Collaborative study to evaluate a reporter gene assay for anti-PD-1 antibody bioactivity / 药学学报

A collaborative inter-laboratory validation was carried out using a reporter gene assay to measure the bioactivity of anti-PD-1 monoclonal antibody, in order to study the applicability and transferability of the method. In this study, two collaborative schemes were designed to measure the precision, linearity and accuracy of the method. The results showed that the 95% confidence interval (CI) of the intra-assay precision was (1.72-16.89) %, inter-assay precision was (2.63-17.67) %, inter-laboratory precision was (9.00-14.26) %, all linear correlation coefficients were greater than 0.99, and the 95% CI for the accuracy at different potency levels was (91.83-104.40) % at 50%, (90.40-101.40) % at 75%, (94.71-105.60) % at 100%, (94.00-102.00) % at 125%, and (96.73-104.30) % at 150%. The collaborative validation results proved that the reporter gene assay for the bioactivity determination of anti-PD-1 monoclonal antibody has good precision, linearity and accuracy, and could be applied to the release and stability analysis of anti-PD-1 monoclonal antibodies in different laboratories.

LC-MS analysis of sintilimab as an anti-PD-1 therapeutic mab for its improved hinge stability study / 药学学报

Monoclonal antibodies (mAbs) have been widely used as therapeutic drugs for treating diseases such as cancers and auto-immune diseases. When using an IgG4 isotype, one of the challenges is the instability of its hinge which is prone to Fab-arm exchange (FAE). The hinge sequence of a wild type IgG4 is -CPSC-, however, a single point mutation S228P from -CPSC- to -CPPC- can effectively diminish FAE, thereby improving hinge stability of the IgG4 molecule. Sintilimab is the fully human anti-PD-1 monoclonal antibody designed and developed for immuno-oncology, in which serine 228 in the hinge was engineered to proline to mitigate FAE. In this study, LC-MS is used to study hinge stability of sintilimab in both in vitro (PBS and human serum) and in vivo (SCID mouse) studies. The studies demonstrate that LC-MS is a fast and simple way to monitor for the occurrence of FAE in vitro and in vivo, and FAE can be eliminated by antibody engineering with a single point mutation.

Effect of electroacupuncture at "Jiaji" (EX-B 2) points combined with nerve mobilization on motor function and mRNA and protein expression of RhoA in rabbits with sciatic nerve injury / 中国针灸

OBJECTIVE@#To observe the effects of electroacupuncture (EA) at "Jiaji" (EX-B 2) points combined with nerve mobilization on protein and mRNA expression of RhoA in rabbits with sciatic nerve injury, and to provide theoretical basis for the treatment of peripheral nerve injury by EA at "Jiaji" (EX-B 2) points combined with nerve mobilization.@*METHODS@#A total of 180 New Zealand rabbits were randomly divided into a normal control group, a model control group, a nerve mobilization group, an EA group, an EA plus nerve mobilization group, 36 rabbits in each group. Each group was further divided into a 1-week subgroup, 2-week subgroup and 4-week subgroup, 12 rabbits in each subgroup. The sciatic nerve injury model was made by clamping method. The rabbits in the normal control group did not receive any intervention. The rabbits in the model control group was normally fed after operation. The rabbits in the nerve mobilization group were treated with nerve mobilization; the manipulation lasted for 1 s and relaxed for 5 s, 10 times per day, 6 days per week. The rabbits in the EA group were treated with EA at "Jiaji" (EX-B 2) points (L-L), once a day, 30 min each time, 6 times per week. The rabbits in the EA plus nerve mobilization group were treated with EA at "Jiaji" (EX-B 2) points, followed by nerve mobilization. The function of sciatic nerve on the injured side was evaluated by toe tension reflex and modified Tarlov score; the tissues of corresponding segments of spinal cord L-L and sciatic nerve were taken; the expression of RhoA gene was detected by real-time PCR and the expression of RhoA protein was detected by Western Blot.@*RESULTS@#① Toe tension reflex and modified Tarlov score: at 1, 2 and 4 weeks, the scores in the model control group were lower than those in the normal control group (all 0.05); at 2 weeks, the expression in the nerve mobilization group was higher than that in the EA group (all <0.01); at 4 weeks, the expression in the nerve mobilization group was lower than that in the EA group (all <0.01).@*CONCLUSION@#The nerve mobilization and EA at "Jiaji" (EX-B 2) points could both promote the repair of injured sciatic nerve, which may be related to the down-regulation of RhoA expression, and the combination of the two methods has better effects.

A study of non-reduced SDS-PAGE purity method of conbercept / 药学学报

A non-reduced SDS-PAGE purity method for quantitation of conbercept fragments was established based on gel screening, comparison of gel imaging system, linearity range of main band, screening of destaining conditions. The results indicated that the bands could be separated effectively with good clearness and flatness on 4%-15% gradient concentration gel, the peaks of all bands could be separated from baseline using high-distinguishability gel imaging system, the signal intensity of a main band had shown a good linearity with ≤ 3 μg of loading amount, and that the destaining was set as a total of ≤ 3 h with exchanging 100 mL destaining buffer every 60 min. The established non-reduced SDS-PAGE method could demonstrate the purity of conbercept more objectively. After validation, the established non-reduced SDS-PAGE method was submitted to FDA in the form of supplementary materials, which laid a quality basis for the direct entry of conbercept to the clinical Ⅲ study in the United States.

Single-site intracorporeal purse-string stitching versus single-port extracorporeal knotting for laparoscopic inguinal inner ring closure in children: a comparative study / 中华普通外科杂志

Objective To compare the surgical and functional outcomes of single-site (transumbilical two-port) intracorporeal purse-suturing (IP) and single-port extracorporeal knotting (EK) for laparoscopic pediatric inguinal hernia (PIH) repair.Methods Between June 2008 and December 2014,358 PIH children underwent lapamscopic inguinal herniorrhaphy,including 126 treated by single-site intracorporeal purse string stitching using a needle-holder (IP group),and 232 by single-port extracorporeal knotting using an inner two-hook needle with preperitoneal hydrodissection (EK group).Results In all patients laparoscopic procedures were completed successfully without conversion.The operating time in IP group was significantly longer than that in EK group [unilateral:(20.4 ± 2.1) min vs.(9.4 ± 1.3) min,t=-5.23,P＜0.01;bilateral:(31.3 ±2.9) min vs.(15.2±1.7) min,t=-4.22,P＜0.01)].The hospital stay were similar between the two groups [(2.35 ±0.25) d vs.(2.38 ±0.18) d,t =-0.062,P ＞ 0.05].Five cases had intraoperative hematoma in the IP group while none in the EK group.One each suffered from recurrence in IP group and EK group.Three postoperative hydroceles were seen in IP group and one in EK group.Subcutaneous knot granulomas were seen in two in EK group.Conclusions Both IP and EK laparoscopic procedures are safe and feasible.With the assistance of preperitoneal hydrodissection technique,single-port laparoscopic EK herniorraphy is superior to single-site IP repair in easy performance and shorter operation time.

Comparison of Pattern-pulse and Pattern-reversal Multifocal Visual Evoked Potentials in Normal Individuals / 法医学杂志

OBJECTIVE@#To compare pattern-pulse multifocal visual evoked potential (PPmfVEP) with pattern-reversal multifocal visual evoked potential (PRmfVEP), and to investigate the symmetry of mfVEP between both eyes in normal individuals.@*METHODS@#The multifocal Vision Monitor was used to observe the mfVEP. T-test and ANOVA were used to analyze P1 wave, amplitude and signal noise ratios (SNR) of two mfVEPs.@*RESULTS@#The SNR and the P1 amplitude reached the maximum at the central visual field and decreased with the increase of eccentricity, and then decreased slowly. The amplitude of the PPmfVEP was significantly smaller than the PRmfVEP in the central retina, while in the peripheral retina the result was exactly the opposite. SNR and amplitude of the PRmfVEP showed no statistical difference in both eyes (P > 0.05). The variance of the amplitude at the same side of visual field was larger than that at the symmetrical visual quadrant.@*CONCLUSION@#mfVEP can reflect the visual function in different parts of retina objectively and exactly. PPmfVEP reflect the vision function of the central retina better than PRmfVEP. The stability of PPmfVEP is better than PRmfVEP in the central retina, while the result is opposite in the peripheral retina. The mfVEP is symmetrical in both eyes of the same individual.

Development of a novel reporter gene method for determination of ADCC potency of anti-CD20 monoclonal antibody / 药学学报

The biological activity of ADCC by anti-CD20 monoclonal antibody was determined by BioGlo™ Luciferase Assay System using Jurkat/NFAT-luc+FcγRIIIa cell line as effector cell and WIL2-S cell line as target cell. The developed method was verified for specificity, precision and accuracy. Anti-CD20 monoclonal antibody showed a dose-response mode by the developed method, and the determination result complied with the following four-parameter equation: y = (A-D)/[1 + (X/C)(B)] + D. The optimized parameters of the method were determined including the antibodies diluted concentration (18,000 ng·mL(-1)), dilution rate (1:5), the ratio of effector cell and target cell (6:1), and induction time (6 h). The values of eight independent tests have passed a statistical test for curve regression analysis, linear or parallelism, which showed the method possessed good specificity. Four different dilute groups of recovery rates sample were determined for 3 times, and the result showed mean relative potencies of (44.39±3.93)%, (72.74±2.78)%, (128.28±7.01)% and (168.19±2.70)% respectively, with a variation coefficient of less than 10%, and the recoveries of (88.78±7.85)%, (96.99±3.70)%, (102.63±5.61)% and (112.12±1.80)% respectively. A novel reporter gene method for determination of biological activity of ADCC by anti-CD20 monoclonal antibody was successfully developed, which showed strong specificity, good reproducibility and high accuracy, and might be used routinely.

Determination of drug antibody ratio in an antibody-drug conjugate / 药学学报

This paper reports the determination of the drug antibody ratio in an antibody-drug conjugate with two methods, i.e. LC-MS and UV/VIS, and to provide a reliable method to scientifically evaluate and effectively control the drug antibody ratio. Deglycosylated sample was analyzed with C4 column followed by MS, and the number of conjugated drugs in the antibody was determined by the molecular weight increase due to the addition of different number of drugs to the antibody, and then drug antibody ratio was calculated by weighted average of different number of drugs conjugated to the antibody. Optical density at 252 and 280 nm was measured with UV/VIS, and due to the difference of extinction coefficients between the antibody and the drug, the drug antibody ratio was calculated from linear equation with two unknowns. The drug antibody ratio was 3.21 and 3.25 respectively measured by the two methods, and the results were similar with the two methods. Our study indicated that both methods, LC-MS and UV/VIS, could be applied to the analysis of drug antibody ratio of the antibody drug conjugate.

The evaluation of SCI by TMS-MEP and its forensic significance / 法医学杂志

OBJECTIVE@#To find an objective and accurate examination for evaluation of spinal cord injury (SCI) in forensic clinical medicine.@*METHODS@#The onset latency of cortex, peak latency of N1, central motor conduction time (CMCT) and wave width of the abductor pollicis brevis and the anterior tibialis were calculated by transcranial magnetic stimulation-motor evoked potential (TMS-MEP). The data of 68 patients suffered from SCI including 23 cervical levels and 45 thoracolumbar levels were collected and compared with that of 30 normal controls.@*RESULTS@#In experimental group, when the muscle strength of the abductor pollicis brevis or the anterior tibialis decreased or disappeared, the onset latency of cortex, the peak latency of N1, and CMCT prolonged and the wave width broadened. And these indexes of grade 2 and 3 muscle strength in experimental group were higher than that in the control group (P < 0.05).@*CONCLUSION@#The TMS-MEP can determine directly and objectively the motor functional status of pyramidal tract of spinal cord in order to provide more accurate and objective evidences in forensic medicine.

Analysis of forensic identification of traumatic macular hole in 16 cases / 法医学杂志

OBJECTIVE@#To analyze and summarize the characteristics of traumatic macular hole and to discuss the key points and matters need attention in forensic identification.@*METHODS@#Sixteen cases of traumatic macular hole were collected from Forensic Science Identification Center of China Medical University from 2004 to 2009 and analyzed.@*RESULTS@#All of the 16 cases resulted from closed ocular contusion. Traumatic macular hole was more common in young men and may occur immediately after injury or after a certain interval. Six months after injury, there was a spontaneous closure of macular hole in 2 cases and the vision of the injured in 12 cases improved to different degrees. According to the degree of vision dysfunction, 12 cases were identified as slight injury, 4 cases were identified as severe injury.@*CONCLUSION@#Traumatic macular hole has its characteristics. Its injury degree is all slight or severe injury. The time of identification should be within 3-6 months.

Analysis of 158 forensic identification cases involved with peripheral nerve injury / 法医学杂志

OBJECTIVE@#To study the characteristics of forensic identification cases involved with peripheral nerve injury, and to discuss how to apply the clinical information, forensic examination and neurophysiological testing.@*METHODS@#One hundred and fifty-eight cases which were diagnosed peripheral nerve injury in clinic, were collected. Then the individual characteristics, injuries, identification results, exaggeration or camouflage were analysed.@*RESULTS@#The male, the unemployed, and the young and middle-aged were common in our cases. The main reasons of "peripheral nerve injury" were traffic accidents and sharp injuries. Most wounded parts were in limbs. Also the exaggeration and camouflage accounted for a considerable proportion in our cases.@*CONCLUSION@#The forensic identification of "peripheral nerve injury" cases should be evaluated with clinical information, forensic examination and electrophysiological testing comprehensively.

Application of spiral CT reconstruction in the forensic identification of atlantoaxial injuries / 法医学杂志

OBJECTIVE@#To evaluate diagnostic value of spiral CT reconstruction in atlantoaxial injuries.@*METHODS@#The images of 25 cases of spiral CT reconstruction were analyzed and compared with images of CT scan and X-ray.@*RESULTS@#In 7 cases of odontoid process fracture, X-ray demonstrated 4 cases and CT demonstrated 5 cases, whereas the spiral CT reconstruction diagnosed 7 cases, which could display the displacement of fracture clearly. The X-ray and CT showed asymmetric space between left and right gaps of atlantoaxial joint in 6 cases, while spiral CT reconstruction showed normal and excluded the possibility of atlantoaxial dislocation. There was one case of lateral atlantoaxial joint dislocation, which was demonstrated by the spiral CT reconstruction clearly but not by the X-ray and CT scan. There were 3 cases of atlantoaxial congenital deformity (1 case of absence of both posterior arch of atlas and odontoid process and 2 cases of maldevelopment of the odontoid process), which were displayed clearly by spiral CT reconstruction, but misdiagnosed as odontoid process fracture and atlantoaxial subluxation by X-ray and CT scan.@*CONCLUSION@#Spiral CT reconstruction can provide the most accurate and integrity imaging information and is very useful in the diagnosis of atlantoaxial injuries and deformity.

The retrospective analysis of 23 cases of post-traumatic abortion / 法医学杂志

OBJECTIVE@#To analyze the clinical characteristics of post-traumatic abortion and the relationship between trauma and abortion.@*METHODS@#We collected and analyzed 23 cases of post-traumatic abortion encountered in the department of clinical forensic medicine, faculty of forensic medicine, China Medical University in recent years.@*RESULTS@#All 23 post-traumatic abort inns were early abortions. Their traumata were mild and external only. Vagina bleeding and abortion occurred shortly after trauma. Most patients were older, from countryside, or unemployed.@*CONCLUSION@#The causes of abortion are complex. Post-traumatic abortion can only be diagnosed after excluding non-traumatic factors. For those cases abortion occur shortly after injuries and non-traumatic factors can not be excluded, trauma should be assessed for its role in abortions as primary, secondary or induction factors correlated with clinical symptoms and pre-

The diagnostic value of SEP in LDH and its forensic significance / 法医学杂志

OBJECTIVE@#To evaluate the diagnostic value of somatosensory evoked potential(SEP) in lumbosacral disc herniation(LDH).@*METHODS@#Posterior tibial nerve somatosensory evoked potential(PTNSEP) and dermatomal somatosensory evoked potential (DSEP) were recorded from 60 patients with radiculopathy caused by LDH and 30 healthy subjects.@*RESULTS@#DSEP was abnormal in 56 cases (93.3%) and PTNSEP was abnormal in 26 cases (43.3%). The significant difference was observed (P<0.001). The abnormality of DSEP from L4 dermatome was detected mainly in patients with L3-4 LDH, the abnormality of DSEP from L5 dermatome was detected mainly in patients with L4-5 LDH, the abnormality of DSEP from L5S1 dermatome was detected mainly in patients with S1 LDH. DSEP can reflect the compressed extent of nerve root.@*CONCLUSIONS@#There was a good correlation of DSEP with lumbosacral nerve root injury,which supplement the information that can not be provided by imaging examingation. DSEP is a sensitive electrophysic method that not only is used to evaluate single nerve root function, but also is helpful in the location of the lesion.