Objective To screen out specific aldosterone(ALD)antibodies using phage display technology and recom-binant antibody technology,providing raw materials for the research and development of ALD diagnostic kits.Methods Five healthy and clean New Zealand white rabbits were selected and immunized for the first time against the diluted ALD-keyhole limpet hemocyanin antigen(2 mg·L-1)using a multi-point injection method on the back,with a dose of 1 mg per rabbit.Immunization was administered again every 2 weeks,with a 50％reduction in dose.Starting from the third immunization,the ear vein blood of the rabbits was collected one week after each immunization.A chemiluminescent plate coated with 0.25 mg·L-1 ALD-bovine serum albumin antigen was used to measure serum titers via indirect and competitive methods.After the 5th immunization,the rabbit with high serum titers and good specificity was selected,and its spleen and bone marrow were removed.The spleen tissue was grinded,and RNA was extracted using TRIzol reagent in one step to obtain gene sequences in the variable region of light chain(VL)and the variable region of heavy chain(VH).The single-chain variable fragment(ScFv)was connected through the linker and constructed into the bacteriophage vector Pcomb3xss;then,it was carried to Escherichia coli TG1 through electrotransformation,and the ALD ScFv phage display library was constructed accordingly.Three to five rounds of enrichment screening were performed against the library.Monoclonal clones,identified by enzyme-linked immunosorbent assay(ELISA)competitive method,were selected for phage supernatant preparation,and a highly competitive clone sequence was obtained.The screened clone sequence was inserted into the pCMV3 expression vector,and the HEK293 cell was transfected using the transient transfection method after the plasmid was extracted.One week later,the supernatant was collected,and its purity and expression were identified by affinity chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE).Results After the 5th immunization,the serum titers of 5 rabbits were indirectly tested,and the results showed that the serum titers of 4# and 5# white rabbits were still greater than 10,000 after being diluted by 32,000 times.The test results based on the competitive method showed that the ratio of low to high values in the plasma sample of 5#white rabbit was 2∶1,superior to that of other white rabbits.The 5# white rabbit was selected for phage library construction.The VL and VH gene fragments were amplified by conventional polymerase chain reaction,and then bridged into ScFv(VL+VH).The agar gel electrophoresis analysis showed that the size of the band was about 750 bp,which was consistent with the size of the originally designed fragment.ScFv was cleaved and electroporated into Escherichia coli TG1 to construct a phage library with a storage capacity of 4.73 × 108 cfu·mL-1.After 3 rounds of washing,300 monoclonal clones were selected from the outbound petri dishes to prepare monoclonal bacteriophages.The ELISA results showed a positive rate of 100％among the 300 clones,and 42 clones were tested positive for calibration competition,with a screening rate of 14％.The 42 positive clones were further subjected to clinical sample competition testing,and 16 monoclonal strains that met the requirements were screened.The 16 strains were retested,and the results of the two tests were consistent.After sequencing,6 antibody sequences were selected for construction and expression.After purification,SDS-PAGE reduced gel electrophoresis results showed that there were bands at positions 50,000 on the heavy chain and 25,000 on the light chain.Six highly affinitive and competitive rabbit ALD monoclonal antibodies were obtained.Conclusion Six highly affinitive and competitive rabbit ALD antibodies are successfully screened using phage display technology,which provides a reference method for the discovery of small molecule antibodies.The screened AD1 85 and AD277 antibodies show a competitive advantage twice that of the positive control in the competition of calibration and clinical samples,providing a possibility for the development of raw materials for ALD detection kits.

AIM:To investigate the mechanism of Piezo1 channel activation promoting the increase in intra-cellular Ca2+ concentration([Ca2+]i)of rat coronary artery smooth muscle cells(CASMCs).METHODS:The primary CASMCs of SD rats were cultured,and the expression and subcellular localization of Piezo1 in the cells were observed by immunofluorescence staining.The Piezo1 and stromal interaction molecule 1(STIM1)in CASMCs were knocked down by siRNA transfection,and the expression levels of the proteins were detected by Western blot.Utilizing laser confocal mi-croscopy,the change of[Ca2+]i in CASMCs was detected by Fluo-4 AM fluorescent probes.RESULTS:It was confirmed by immunofluorescence staining that the expression of Piezo1 existed in primary rat CASMCs.Immunofluorescence staining also showed that Piezo1 was co-located with sarco-/endoplasmic reticulum Ca2+-ATPase 2(SERCA2),mitochondrial outer membrane protein TOM20 and nuclear membrane protein lamin B1.Western blot results showed that the protein expres-sion levels of STIM1 and Piezo1 were significantly down-regulated by siRNA transfection(P<0.05).Compared with con-trol group,Yoda1,the agonist of Piezo1,could increase the extracellular Ca2+ influx of CASMCs(P<0.01).However,the Ca2+ influx mediated by Yoda1 was not affected by the inhibition of L-type calcium channels.Treatment with Yoda1 in-creased the intracellular Ca2+ release of CASMCs(P<0.01).However,inhibition of calcium channels on endoplasmic re-ticulum,ryanodine receptor and inositol 1,4,5-triphosphate receptor,did not affect intracellular Ca2+ release mediated by Yoda1.After the Ca2+ in endoplasmic reticulum was emptied using thapsigargin(TG),Yoda1 also mediated the Ca2+ re-lease of other organelles in CASMCs(P<0.01).After inhibition of L-type calcium channels,treatment with store-operated calcium channel(SOCC)inhibitor BTP2 or knockdown of STIM1 led to the decrease in extracellular Ca2+ influx of CASMCs mediated by Yoda1(P<0.01).Treatment with TG increased the release of Ca2+ from the endoplasmic reticulum of CASMCs after knockdown of Piezo1(P<0.05),but the extracellular Ca2+ influx mediated by TG was not affected.After inhibition of L-type calcium channels and SOCC,knockdown of Piezo1 led to the decreases in intracellular Ca2+ release and extracellular Ca2+ influx induced by Yoda1(P<0.01).CONCLUSION:The Piezo1 agonist orchestrates the influx of extracellular Ca2+ by activating Piezo1 channels on the cell membrane and inducing the indirect activation of SOCC.More-over,it facilitates the release of Ca2+ from organelles.Consequently,these pathways synergistically elevate the[Ca2+]i of rat CASMCs.

AIM:To investigate the influence of fat mass and obesity-associated(Fto)gene on the aberrant contraction of aortic smooth muscle in diabetes mellitus(DM)mice,and to explore the mechanism of Fto gene underlying the calcium regulation.METHODS:Smooth muscle-specific Fto gene knockout(FtoSMKO)mice were generated using Cre-loxP technology.The experiment involved 3 groups of mice:wild-type(WT)group,DM model group and FtoSMKO-DM group,with 15 mice in each group.In DM group and FtoSMKO-DM group,type 1 DM was induced by intraperitoneal injec-tion of streptozotocin.The mice in WT group were injected with equal volume of citric acid-sodium citrate buffer solution.The influences of different drugs on the contraction responses of aortic smooth muscle in mice were analyzed using a multi-myograph system.The expression level of FTO protein in the aortic tissues was detected by Western blot.RESULTS:(1)Compared with WT mice,the expression levels of FTO protein in the aortic tissues of DM mice were significantly in-creased(P＜0.01).(2)The expression level of FTO protein in smooth muscle was significantly decreased after knockout of Fto gene(P＜0.01).Compared with WT group,the mice in DM group exhibited a significant decrease in body weight and a marked increase in fasting blood glucose level(P＜0.05).There were no noticeable differences in body weight or fasting blood glucose level between FtoSMKO-DM group and DM group(P＞0.05).(3)The contraction responses of aortic smooth muscle in DM group were substantially increased by phenylephrine compared with WT group.Specifically,vaso-constriction responses mediated by non-L-type calcium channels and store-operated calcium channels(SOCC)were signifi-cantly enhanced in DM group.In addition,the responses mediated by inositol 1,4,5-trisphosphate receptors(IP3R),which facilitate calcium release from the sarcoplasmic reticulum,were significantly enhanced.However,the responses mediated by caffeine-activated ryanodine receptors(RyR),which also facilitate calcium release from the sarcoplasmic re-ticulum,were significantly inhibited(P＜0.05).(4)Compared with DM group,the phenylephrine-induced contraction re-sponses of aortic smooth muscle in FtoSMKO-DM group were greatly weakened(P＜0.05).In particular,the vasoconstriction responses mediated by non-L-type calcium channels and SOCC in FtoSMKO-DM group were greatly suppressed(P＜0.05),while those mediated by caffeine-activated RyR were dramatically boosted(P＜0.05).However,IP3R-mediated responses were not affected(P＞0.05).CONCLUSION:Smooth muscle-specific Fto gene knockout suppresses contractile hyperre-sponsiveness in the aortic smooth muscle of DM mice,which may be attributed to involvement of FTO protein in calcium regulation in the vascular smooth muscle.

Objective To study the effects of different pellet feed hardness on the growth and reproduction,feed utilization rate,and environmental dust in laboratory mice.Methods One hundred of fifty 50 3-week-old SPF-grade C57BL/6JGpt and 150 ICR laboratory mice were randomly divided into three groups,with an equal number of males and females.They were fed diets with different hardness of 18.62 kg,23.15 kg,and 27.89 kg.Body weight,feed utilization rate,and dust levels in cages were recorded and calculated for mice aged 3-10 weeks.Forty-five 6-week-old male mice and ninety 4-week-old female mice from each strain were randomly divided into three groups and fed pellet feeds with three different hardness levels.After 2 weeks of adaptation to the same hardness feed,the mice were paired at a 1:2 male-to-female ratio and monitored for reproductive data for 3 months.Results At the age of 4 weeks,the body weight of male C57BL/6JGpt mice in 23.15 kg group was significantly higher than that in the 18.62 kg and 27.89 kg groups(P＜0.01),and the body weight of females in the 18.62 kg group was significantly higher than that in the 27.89 kg group(P＜0.05).There was no significant difference in body weight among ICR mice aged 3-10 weeks across different feed hardness groups(P＞0.05).For both strains,feed utilization rate for males was higher than that for females across different feed hardness groups at all weeks of age(P＜0.01).Compared to the 27.89 kg group,both the 18.62 kg and 23.15 kg groups showed a significant increase in the 50-mesh dust levels in cages for both strains aged 4-8 weeks(except for 7-week-old C57BL/6JGpt mice)(P＜0.05).For both C57BL/6JGpt and ICR mice,there was no significant difference in basic reproductive performance such as interval between the first litter and the monthly production index among the three feed hardness groups during the experimental period(P＞0.05).However,the monthly production index of C57BL/6JGpt mice first increased and then decreased with the increase of feed hardness,while that of ICR mice increased with increasing feed hardness,though these differences were not statistically significant(P＞0.05).Conclusion Different strains and genders had different tolerance to feed hardness.C57BL/6JGpt mice are more adapted to lower hardness feeds,while ICR mice are better suited to slightly higher hardness feeds.

Objective:To explore the clinical and genetic characteristics of two children with mental retardation and microcephaly.Methods:Two children who had visited the Anhui Children′s Hospital respectively on March 12 and June 22, 2021 were selected as the study subjects. Peripheral venous blood samples were collected from them and their parents, and subjected to chromosomal karyotyping and whole exome sequencing analyses. Candidate variants were verified by Sanger sequencing and pathogenicity analysis. This study was approved by the Anhui Children′s Hospital (Ethics No. EYLL-2018-008).Results:Chromosomal karyotyping and copy number detection of the two children had found no abnormality. Whole exome sequencing revealed that child 1 has harbored a c. 471delT (p.Pro157Profs*9) frameshifting variant of the CASK gene, whilst child 2 has harbored a c. 1259_1269delCTGAGAATAAC (p.Pro420fs*27) frameshifting variant of the CASK gene. Sanger sequencing confirmed that both variants were de novo in origin. Based on the guidelines from the American College of Medical Genetics and Genomics and the Association for Molecular Pathology (ACMG-AMP), both variants were rated as pathogenic (PVS1+ PS2+ PP3). Conclusion:The de novo variants of the CASK gene probably underlay the pathogenesis of mental retardation and microcephaly in both children.

Objective:To investigate the prevalence and related influencing factors of thyroid nodules in different occupational groups in Shijiazhuang City.Methods:Different occupational groups who underwent physical examination at the Second Hospital of Shijiazhuang City from May to December 2015 and had lived there for more than 10 years were selected as the investigation subjects, including workers, farmers, science and technology education personnel, medical and health personnel, civil servants and individual businessmen. Questionnaire survey and thyroid examination were conducted, blood lipid, blood glucose and thyroid hormone levels were tested, and related influencing factors of thyroid nodules were analyzed.Results:A total of 1 440 different occupational subjects were surveyed, and the prevalence of thyroid nodules was 20.35% (293/1 440). The prevalence of thyroid nodules in females (22.59%, 159/704) was significantly higher than that in males (18.21%, 134/736), and the difference was statistically significant (χ 2 = 4.26, P = 0.039). There was a statistically significant difference in the prevalence of thyroid nodules in different age groups (χ 2 = 73.87, P < 0.001), with the highest prevalence in the 70 - 80 age group (43.75%, 21/48). There was a statistically significant difference in the prevalence of thyroid nodules among different occupational groups (χ 2 = 36.56, P < 0.001), with the highest prevalence in individual businessmen (31.84%, 78/245). By univariate analysis, the prevalence of thyroid nodules in people with good economic status [27.45% (42/153) vs 19.48% (248/1 273)], radiation exposure history [26.39% (109/413) vs 17.92% (184/1 027)], and diabetes history [33.71% (30/89) vs 19.47% (263/1 351)] was higher (χ 2 = 5.35, 13.06, 10.45, P < 0.05). Conclusions:The prevalence of thyroid nodules in occupational group in Shijiazhuang City is relatively high, and the prevalence of thyroid nodules in females is significantly higher than that in males. Economic status, radiation exposure history and diabetes history are the main influencing factors.

Objective To construct and apply a management plan for microaspiration of oropharyngeal secretions in ICU intubated patients.Methods Based on evidence summaries and expert consultation,a management plan for microaspiration of oropharyngeal secretions in ICU intubated patients was constructed,consisting of 19 items covering 7 aspects including identification of risk factors,position management,tube and cuff selection,cuff management,mechanical ventilation management,pain and sedation management,removal of oropharyngeal and subglottic secretions,and oral care.Convenience sampling was used to select 141 ICU intubated patients from a tertiary A comprehensive hospital in Suzhou from June,2022 to September,2023.Patients were divided into an experimental group(n=72)and a control group(n=69)according to the wards.The experimental group received the management plan for microaspiration of oropharyngeal secretions in ICU intubated patients.The control group received the nursing bundle for ventilator associated pneumonia(VAP).The incidence and time from intubation to microaspiration and VAP,duration of mechanical ventilation,ICU length of stay,and disease outcome were compared between the 2 groups.Results The incidence of microaspiration of oropharyngeal secretions,the duration of mechanical ventilation,time from intubation to microaspiration showed significant differences between the 2 groups(P<0.05).There were no significant differences in the incidence of ventilator associated pneumonia,ICU length of stay,and disease outcome between the 2 groups(P>0.05).The time from intubation to VAP in the experimental group was 7.5 days,and that in the control group was 3.8 days.Conclusion The application of the management plan for microaspiration of orophaiyngeal secretions in ICU intubated patients is beneficial for reducing the incidence of microaspiration,delaying the time from intubation to microaspiration and VAP,and shortening the duration of mechanical ventilation.

Objective:To retrieve, evaluate, and integrate the best practice evidence for targeted temperature management in neurocritically ill patients.Methods:PIPOST tool was used to identify evidence-based issues; the Chinese and English terms were chosen as "Traumatic brain injuries", "Craniocerebral trauma/Cerebral hemorrhage/Stroke/Subarachnoid hemorrhage/Cerebral vascular disease", and "Mild hypothermia therapy/Target body temperature/Cyotherapy". According to top-down design of the "6S" evidence model, databases such as French guideline network, US guideline network, UpToDate, Chinese Biomedical Literature Database, China Yimaitong Guide network, CNKI, and Wanfang Medical Network were searched. Two researchers independently evaluated the quality of the included literature, and extracted and summarized the evidences of the literature that met the quality standards.Results:A total of 10 articles were included, including 1 clinical decision, 4 guidelines, 1 evidence summary, and 4 expert consensuses. Finally, 27 pieces of best evidences were summarized, including 6 aspects as evaluation, preparation before implementation, targeted body temperature selection, intervention duration, chill management, and other complication prevention.Conclusion:Best evidence for temperature management in neurocritically ill patients can provide evidence-based evidence for healthcare professionals.

To analyze the correlation between the duration of hyperbaric oxygen (HBO) therapy and oral medication clonazepam reduction in patients with paroxysmal sympathetic hyperactivity (PSH).

ObjectiveThis study aimed to investigate the HIV genotypic subtypes and molecular transmission clusters among men who have sex with men （MSM） with newly reported HIV infections in Dehong Dai and Jingpo Autonomous Prefecture （Dehong Prefecture）， Yunnan Province， China， between 2010 and 2019. The study aimed to identify potential high-risk transmitters and provide reference data for screening， management， and intervention of infection sources. MethodsPlasma samples from newly reported HIV-positive MSM individuals in Dehong Prefecture between 2010 and 2019 were collected. The viral pol gene fragments were amplified， sequenced， and genotyped. Genetic distances （GD） between pairwise sequences were analyzed and calculated. MEGA 7.0 and Gephi were used for phylogenetic and molecular transmission network analysis. ResultsA total of 159 newly reported HIV infections among MSM were included in the study， with successful genotyping of 100 cases. Nine HIV-1 subtypes were identified， with the most prevalent being CRF01_AE subtype （52%）， followed by CRF07_BC subtype （31%）， CRF55_01B subtype （10%）， and others （7%）. Cluster analysis revealed a total network access rate of 67%， forming three transmission clusters. CRF01_AE subtype formed two transmission clusters with 38 and 3 infected individuals， while CRF07_BC subtypes formed one transmission cluster with 26 infected individuals. The transmission network within the CRF01_AE clusters exhibited a more complex relationship. Significant differences in educational level were observed between the two main transmission clusters. ConclusionThe predominant HIV subtypes among newly reported MSM cases in Dehong Prefecture between 2010 and 2019 were CRF01_AE and CRF07_BC. Significant cultural differences are observed between the main transmission clusters. Continued monitoring of genotypic subtypes and targeted interventions within transmission clusters are warranted.