This study is designed to address the development, synthesis, and screening of non-animal-derived nanoantibody libraries. Furthermore, it seeks to elucidate the impact of framework region selection and complementarity-determining region (CDR) design on the characteristics of synthesized nanoantibody libraries. These investigations aim to establish a robust theoretical and technical foundation for enhancing the efficacy, diversity, and practical applicability of synthetic nanoantibody libraries. In this study, a new framework (IGHV3S65*01-IGHJ4*01) was identified based on the high-throughput sequencing results of natural nanobodies, and degenerate primers were designed based on the frequency of amino acids at each position in the complementarity-determining region (CDR) region to synthesize the coding fragments of nanobodies by overlap PCR. After 40 times of electro-transformation, a single-frame synthesized nanobody library (SS-Library) containing 6×10⁹ clones was obtained, and the titer of the library was demonstrated to be 10¹³ PFU/mL after rescue by the helper phage M13K07. Random 48 single colonies were picked for PCR, which revealed an insertion rate of 95.8%. Sanger sequencing results showed that 38 clones had complete sequences, none of which showed cysteines or stop codons, and no identical sequences appeared, suggesting that the library had higher diversity. The library was screened and validated with three antigens, including bovine serum albumin (BSA), acetylcholinesterase (AchE), and immunoglobulin G (IgG). Finally, 2 nanobodies against BSA, 10 against AchE, and 15 against IgG were obtained. One positive clone of each antigen was singled out for recombinant expression, and the results showed that all the three nanobodies were expressed in a soluble form. The binding activity of recombinantly expressed nanobodies was evaluated using indirect enzyme-linked immunosorbent assay (ELISA) and bio-layer interferometry (BLI). The results demonstrated that the anti-AChE and anti-IgG nanobodies exhibited specific binding to their respective antigens, with affinity constants (KD) of 294 nmol/L and 250 nmol/L, respectively. The nanobody synthetic library preparation method proposed in this study is simple and easy to use with low preference, and it is expected to be a universal nanobody discovery platform for the preparation and development of lead specific nanobodies.
Single-Domain Antibodies/biosynthesis* ; Peptide Library ; Complementarity Determining Regions/immunology* ; Animals

Objective:To investigate and compare the regulatory effects of umbilical cord mesenchymal stem cells (MSC) and their conditioned medium (MSC-CM) on gut microbiota of septic mice.Methods:Twenty-eight six-to-eight-week-old female C57BL/6J mice were randomly divided into sham operation group (Sham group), sepsis model group (CLP group), sepsis+MSC treatment group (CLP+MSC group) and sepsis+MSC-CM treatment group (CLP+MSC-CM group), with seven mice in each group. The septic mouse model was established by cecal ligation and puncture (CLP). In Sham group, CLP were not performed, and other operations were the same as CLP group. Mice in the CLP+MSC group and CLP+MSC-CM group received 0.2 mL 1×10 6 MSC or 0.2 mL concentrated MSC-CM via intraperitoneal injection 6 hours after CLP, respectively. Sham group and CLP group were given 0.2 mL sterile phosphate buffer saline (PBS) via intraperitoneal injection. Histopathological changes were evaluated by hematoxylin-eosin (HE) staining and colon length. Levels of inflammatory factors in serum were detected by enzyme-linked immunosorbent assay (ELISA). Phenotype of peritoneal macrophages was analyzed by flow cytometry, and the gut microbiota was analyzed via 16S rRNA sequencing. Results:Compared with Sham group, significant inflammatory injury in lung and colon was observed, and shorter colon was detected in CLP group (cm: 6.00±0.26 vs. 7.11±0.09), the level of inflammatory cytokine interleukin-1β (IL-1β) in serum was significantly increased (ng/L: 432.70±17.68 vs. 353.70±17.01), the proportion of F4/80 + peritoneal macrophages was increased [(68.25±3.41)% vs. (50.84±4.98)%], while the ratio of F4/80 +CD206 + anti-inflammatory peritoneal macrophages was decreased [(45.25±6.75)% vs. (66.66±3.36)%]. The α diversity sobs index of gut microbiota was downregulated significantly (118.50±23.25 vs. 255.70±6.87), the structure of species composition was altered, and the relative abundance of functional gut microbiota related to transcription, secondary metabolites biosynthesis, transport and catabolism, carbohydrate transport and metabolism, and signal transduction were decreased significantly in CLP group (all P < 0.05). Compared with CLP group, upon MSC or MSC-CM treatment, the pathological injury in lung and colon was alleviated to varying extent, the length of colon was increased (cm: 6.53±0.27, 6.87±0.18 vs. 6.00±0.26), the level of IL-1β in serum was downregulated (ng/L: 382.10±16.93, 343.20±23.61 vs. 432.70±17.68), the ratio of F4/80 + peritoneal macrophages was decreased [(47.65±3.93)%, (48.68±2.51)% vs. (68.25±3.41)%], the ratio of F4/80 +CD206 + anti-inflammatory peritoneal macrophages was increased [(52.73±5.02)%, (66.38±4.73)% vs. (45.25±6.75)%], and the α diversity sobs index of gut microbiota was increased (182.50±16.35, 214.00±31.18 vs. 118.50±23.25), and the effects of MSC-CM were more significant (all P < 0.05). At the same time, species composition of gut microbiota was rebuilt, and a tendency of increase in relative abundance of functional gut microbiota was observed upon MSC and MSC-CM treatment. Conclusion:Both MSC and MSC-CM could alleviate inflammatory injury in tissues, and showed regulatory effects on gut microbiota in septic mouse model, moreover, MSC-CM exhibited superior advantages over MSC.

The present study developed an ultra-fast liquid chromatography coupled with triple quadrupole-linear ion trap composite mass spectrometry(UHPLC-QTRAP-MS) to simultaneously determine the content of potential active components in Scutellariae Barbatae Herba and also to provide a reference approach for screening out the differential quality control components among different batches of Scutellariae Barbatae Herba. Chromatographic separations were conducted on a Thermo Acclaim~(TM) RSLC 120 C_(18) column(3.0 mm×100 mm, 2.2 μm) in a gradient program. The mobile phase consisted of 0.1% aqueous formic acid and acetonitrile, and the column temperature was maintained at 40 ℃. The flow rate was 0.4 mL·min~(-1) and the injection volume was 2 μL. The targeted compounds were monitored in the multiple reaction monitoring(MRM) mode. The acquired data were processed by hierarchical cluster analysis(HCA) and partial least square discriminant analysis(PLS-DA). Sixteen compounds all showed good linear relationship within the corresponding linear ranges and the R~2 values were all higher than 0.993 2. The RSDs of precision, repeatability, and stability were less than or equal to 3.7%. Mean recovery rates were in the range of 95.67% and 104.8% with RSDs≤3.2%. According to HCA and PLS-DA, all samples were clustered into four categories. Scutellarin, acteoside, scutellarein, and scutebarbatine X(VIP>1) were considered as differential chemical markers in the four categories. In conclusion, the developed method can be used for the simulta-neous determination of the multiple components and quality control of Scutellariae Barbatae Herba.
Chemometrics ; Chromatography, High Pressure Liquid/methods* ; Chromatography, Liquid ; Scutellaria ; Tandem Mass Spectrometry/methods*

Objective:To compare the therapeutic effects and safety of dexmedetomidine and midazolam on patients with severe coronavirus disease 2019 (COVID-19) who received non-invasive ventilation.Methods:Patients with COVID-19 who needed non-invasive ventilation in one critical care medicine ward of Wuhan Jinyintan Hospital during the team support period from the department of critical care medicine of Renmin Hospital of Wuhan University from January 23rd to February 15th in 2020 were investigated retrospectively. Ramsay score, mean arterial pressure (MAP), heart rate (HR), respiratory rate (RR), arterial oxygen partial pressure (PaO 2) before sedation and at 1, 12, 24 hours after sedation, sleep time were collected, and the side effects such as excessive sedation, fall of tongue, abdominal distension, aspiration, bradycardia, escalation to invasive mechanical ventilation during 24 hours were also collected. According to different sedative drugs, patients were divided into the control group (without sedative drugs), dexmedetomidine group and midazolam group. The changes of indicators among the three groups were compared. Results:Fourteen patients were injected with dexmedetomidine (loading dose of 1 μg/kg for 10 minutes, maintained at 0.2-0.7 μg·kg -1·h -1); 9 patients were injected with midazolam (loading dose of 0.05 mg/kg for 2 minutes, maintained at 0.02-0.10 mg·kg -1·h -1); 12 patients didn't use sedative drugs due to limitations of previous hospital or patients' rejection. In dexmedetomidine group and midazolam group, the Ramsay score was maintained at 2-3 points after sedation, which were higher than those of control group at different time points after sedation, and there was no significant difference between dexmedetomidine group and midazolam group. MAP of dexmedetomidine group and midazolam group decreased gradually after sedation. MAP after 1-hour sedation was significantly lower than that before sedation, and MAP after 24 hours sedation was significantly lower than that in the control group [mmHg (1 mmHg = 0.133 kPa): 109.7±11.5, 107.1±12.3 vs. 121.1±13.3, both P < 0.05]. HR decreased gradually after sedation treatment, which was significantly lower after 12 hours of sedation than that before sedation, and HR in dexmedetomidine group was significantly lower than that in control group after 12 hours of sedation (bpm: 84.0±13.9 vs. 92.8±15.4 at 12 hours; 81.0±16.7 vs 92.6±12.7 at 24 hours, both P < 0.05). PaO 2 increased and RR decreased in all three groups after ventilation. PaO 2 in dexmedetomidine group and midazolam group were significantly higher than that in the control group after 12 hours of sedation [cmH 2O (1 cmH 2O = 0.098 kPa): 79.0±6.5, 79.0±8.9 vs. 70.0±7.8, both P < 0.05]; the decreases of RR in dexmedetomidine group and midazolam group were significant than that in control group after 1 hour of sedation (bpm: 34.0±3.9, 33.8±4.6 vs. 39.0±3.6, both P < 0.05). There were no differences of MAP, HR, PaO 2 and RR between dexmedetomidine group and midazolam group at different time points. The sleep duration in dexmedetomidine group and midazolam group were significantly longer than that in the control group (hours: 4.9±1.9, 5.8±2.4 vs. 3.0±1.8, both P < 0.05), but there was no difference between dexmedetomidine group and midazolam group ( P > 0.05). Adverse events occurred in all three groups. In midazolam group, there were 2 cases of excessive sedation with fall of tongue and abdominal distension, including 1 case of aspiration, 1 case receiving intubation due to refractory hypoxemia and 1 case due to unconsciousness. In dexmedetomidine group, there were 2 cases of bradycardia, 1 case of intubation due to refractory hypoxemia. In control group, 4 cases underwent intubation due to refractory hypoxemia. Conclusions:Non-invasive mechanical ventilation is an important respiratory support technology for patients with severe COVID-19. Appropriate sedation can increase the efficiency of non-invasive mechanical ventilation. Dexmedetomidine is more effective and safer than midazolam in these patients, but attention should be paid to HR and blood pressure monitoring.

Objective To compare the efficacy of operative and non-operative methods in the treatment of severe thoracic trauma in Tibetan Plateau.Methods A retrospective case-control study was conducted to analyze 286 patients with severe thoracic trauma admitted to the Shigatse People's Hospital from August 2016 to October 2018.There were 206 males and 80 females,aged 13-71 years [(34.3 ±11.6) years].The duration from injury to hospital ranged from 2 to 49 hours [(22.8 ± 8.3) hours].The causes of injury including fall from height in 109 patients,traffic injury in 98,crush injury in 32,blunt injury in 29,cattle head injury in 9,knife stab injury in 6 and other causes in 3.The injury severity score (ISS) varied from 16 to 48 points on admission [(24.2 ± 8.8) points].A total of 159 patients underwent operation (Operation group) and 127 patients underwent non-operative treatment (Non-operation group).The ISS score was (25.2 ± 8.3)points in Operation group and (23.7 ±7.9)points in Non-operation group.The length of hospital stay,intensive care unit (ICU) care time,complication rate and mortality were recorded in two groups.The correlation of ISS and age with mortality was investigated.Results The length of hospital stay was (12.2 ± 3.8) days in the Operation group and (19.7 ± 5.8) days in the Non-operation group (P ＜ 0.05).In the Operation group,27 patients were admitted to the ICU and stayed for (4.7 ± 1.3)days,and in the Non-operation group 33 patients were admitted to the ICU and stayed for (11.7 ± 3.2) days (P ＜ 0.05).The complication rate was 17.6％ in the Operation group and 31.5％ in the Non-operation group (P ＜0.05).Two patients died in the Operation group,with the mortality rate of 1.3％;six patients died in the Non-operation group,with the mortality rate of 4.7％,with no statistical difference (P ＞ 0.05).There were statistically significant differences in the age of death between the Operation group and the Non-operation group (P ＜ 0.05).The differences in the ISS score and age between the survival and death patients within the two groups were statistically significant (P ＜0.05).Logistic regression analysis showed age (OR =1.090,95％ CI 1.002-1.186) and ISS (OR =1.058,95％ CI 1.027-1.090) were slqnificantly related to mortality.Conclusions For severe thoracic trauma in Tibetan Plateau,operative treatment can shorten the length of hospital stay and ICU care time and reduce the incidence of complications.The mortality ratio of the two groups has no significant difference.Age and ISS may be the high risk factors for death of severe thoracic trauma patients.

OBJECTIVE: To investigate the efficacy of intraoperative cognitive navigation on laparoscopic radical prostatectomy using 3D prostatic models created by U-shaped convolutional neural network (U-net) and reconstructed through Medical Image Interaction Tool Kit (MITK) platform. METHODS: A total of 5 000 pieces of prostate cancer magnetic resonance (MR) imaging discovery sets with manual annotations were used to train a modified U-net, and a set of clinically demand-oriented, stable and efficient full convolutional neural network algorithm was constructed. The MR images were cropped and segmented automatically by using modified U-net, and the segmentation data were automatically reconstructed using MITK platform according to our own protocols. The modeling data were output as STL format, and the prostate models were simultaneously displayed on an android tablet during the operation to help achieving cognitive navigation. RESULTS: Based on original U-net architecture, we established a modified U-net from a 201-case MR imaging training set. The network performance was tested and compared with human segmentations and other segmentation networks by using one certain testing data set. Auto segmentation of multi-structures (such as prostate, prostate tumors, seminal vesicles, rectus, neurovascular bundles and dorsal venous complex) were successfully achieved. Secondary automatic 3D reconstruction had been carried out through MITK platform. During the surgery, 3D models of prostatic area were simultaneously displayed on an android tablet, and the cognitive navigation was successfully achieved. Intra-operation organ visualization demonstrated the structural relationships among the key structures in great detail and the degree of tumor invasion was visualized directly. CONCLUSION The modified U-net was able to achieve automatic segmentations of important structures of prostate area. Secondary 3D model reconstruction and demonstration could provide intraoperative visualization of vital structures of prostate area, which could help achieve cognitive fusion navigation for surgeons. The application of these techniques could finally reduce positive surgical margin rates, and may improve the efficacy and oncological outcomes of laparoscopic prostatectomy.
Humans ; Laparoscopy ; Magnetic Resonance Imaging ; Male ; Neural Networks, Computer ; Prostate ; Prostatectomy

Objective To investigate the safety of intraoperative electron radiation therapy (IOERT) for stage Ⅰ hepatocellular carcinoma (HCC) by a cohort study.Methods From November 2010 to May 2012,16 patients who were pathologically diagnosed with stage Ⅰ HCC underwent IOERT after radical resection.With a cohort study,87 patients with stage Ⅰ HCC who underwent radical resection alone during the same period were qualified,and according to tumor size (＞ 5 cm and ≤ 5 cm) and resection margin (close margin and negative margin),32 of 87 patients made up the control group.The intraoperative and postoperative adverse events,liver function parameters,coagulogram,and routine blood parameters,as well as IOERT-related adverse reactions,were evaluated.Independent-samples t test was used for analyzing the differences between groups.Results Compared with the control group,the IOERT group had a significantly longer operative time ((275.4 ± 71.55) min vs.(184.7 ± 64.74) min,P =0.000),a slightly higher incidence of intraoperative adverse events (18.75％ vs.6.25％,P=1.000),a slightly lower incidence of operative complications (12.50％ vs.28.12％,P =0.460),and a lower perioperative mortality (0 vs.6％,P =0.440).Liver function parameters showed no significant differences between the two groups (P ＞ 0.05).There were no significant differences between the two groups in postoperative time to grade 1 or normal liver function parameters,median length of postoperative hospital stay,length of hospital stay in the surgical department,time to incision healing,and level of incision healing (P ＞ 0.05).During follow-up,no radiation hepatitis was found in the IOERT group.Conclusions As an adjuvant therapy after radical resection for early HCC,IOERT has no significant side effects on postoperative recovery and liver function,and an intraoperative dose of 15-16 Gy is safe.

AlM: To measure ocular biometric values with the degree of axial myopia and determine the relationship between the differences using the Lenstar 900.
METHODS:Totally 413 myopes (826 eyes) were enrolled in this study and were divided into 3 groups: low myopia (myopia <3. 00 diopters, 104 eyes), moderate myopia ( myopia =3. 00-6. 00 diopters, 500 eyes) and high myopia ( myopia > 6. 00 diopters, 222 eyes ). Central corneal thickness ( CCT ) , aqueous depth ( AD ) , lens thickness (LT), axial length (AL) were measured by Lenstar 900. The parameters were tested using analysis of variance and the relations among SE, AL, LT were analyzed.
RESULTS: There were significant difference both in AL (F=206.16, P<0.01) and AD (F=4.764, P<0.05) and no significant difference both in CCT and LT between these myopia groups. With analysis of Person, the Spherical equivalent ( SE) shows a significantly positive correlation with AL (r=0. 662, P<0. 01) and AD (r=0. 095, P<0. 05), no correlation with CCT and LT. AL shows a significantly positive correlation with AD (r=0. 347, P<0. 01) and CCT (r=0.126, P<0.01), negative correlation with LT (r=-0.265, P<0. 01). LT shows a significantly negative correlation with AD (r=-0. 496, P<0. 01).
CONCLUSlON: Along with the diopters increasing in myopia, the axial length and aqueous depth continue to increase. Spherical equivalent ( SE) shows a significantly positive correlation with AD. AL shows positive correlation with SE, AD and CCT and negative correlation with LT. LT shows a significantly negative correlation with AD.

OBJECTIVETo establish homozygous transgenic mouse strain expressing human tau isoform with P301L mutation.

METHODSFive transgenic mice expressing human tau isoform with P301L mutation were obtained by microinjection into male nuclei. Homozygote and hemizygote were identified by PCR and real-time fluorescent quantitative PCR.

RESULTSNinety five homozygous transgenic mice were selected, and the results indicated that homozygous transgenic mice were superior to hemizygote in simulating the changes of biological characteristics.

CONCLUSIONExogenous gene tau is able to stably transmit to next generation and the combination of SYBR Green real-time fluorescent quantitative PCR with the traditional mating is a fast, reliable and economical way to screen homozygous and hemizygous transgenic mice.

Animals ; Female ; Homozygote ; Humans ; Male ; Mice ; Mice, Inbred C57BL ; Mice, Transgenic ; Microinjections ; Mutation ; tau Proteins ; genetics

Objective To design a wireless transmission solution of multi-parameter vital signs signal which can be applied in psychological status assessment.Methods C8051F350 SOC was used to define data frame structure,on application level,including channel number,control words,data and so on,which put all nodes data into the upper computer cache area by channel in an orderly way.A transfer rate of five seconds on a grid,each grid at least 48 groups of data was chosen in buffer,which could not only match the dynamic PC image display,but also to achieve the stability of the channel signal acquisition and orderly transfer.Results After operating test,with the communication distance within 10～15 m,the dynamical display of the upper computer showed a smooth curve data,which met the system design requirement.After optimization of the data frame,the transmission power could be reduced to 1/12 of that of normal mode.The whole system with three advantages of small size,low power and easy to carry.Conclusion Multi-parameter wireless acquisition system described in this paper eliminates the data line connected to the subjects,which does not affect their normal activities and is conducive to the real response of subjects' mental state,and provides a reliable means of detection for psychological assessment.