Programmed cell death protein ligand-1(PD-L1)is an important immune checkpoint molecule that plays an important role in regulating the body's immune response.Several clinical studies have shown that the expression level of PD-L1 in tumors is closely related to the efficacy of immune checkpoint inhibitors.Due to the spatial and temporal heterogeneity of tumors,immunohistochemical methods commonly used in clinical practice cannot accurately and comprehensively reflect the ex-pression level of PD-L1 in patients.Given that nuclear-medicine molecular imaging technology can noninvasively,real-time,dy-namically and visually monitor the expression level of PD-L1 in vivo at the molecular level,this article mainly focuses on the re-search of peptide-based radiolabeled molecular probes targeting PD-L1,with the aim of providing guidance for the search of no-vel peptide molecular probes for immunoimaging as well as for the screening of immunotherapy-suitable patients and evaluation of therapeutic efficacy,and other clinical applications.

Objective A HPLC-quantitative analysis of multi-components by single marker(QAMS)was established to determine 5 ingredients including uracil,uridine,hypoxanthine,inosine and guanosine in Periplaneta americana.Methods Separation took place on a Agilent ZORBAX SB-Aq column(250 mm×4.6 mm,5 μm)by gradient elution of methanol-0.01 mol·L-1 potassium dihydrogen phosphate at 20℃with a flow rate of 1.0 mL·min-1.The detection wavelength was 260 nm and the injection amount was 10 μL.The relative correction factors(fa/b)was calculated for the other four components with uridine as an internal standard.The content of 5 ingredients in 10 batches of Periplaneta americana was determined by QAMS.Results were compared with those of external standard method(ESM).Results Five nucleosides showed good linear relationships in their own ranges(r＞0.999 5),and the average recoveries ranged from 97.0%to 100.8%.The relative correction factors of uracil,hypoxanthine,inosine and guanosine were 0.908 0,1.005 3,1.969 5 and 1.303 4,respectively.Conclusion The established method is accurate and stable.It can provide theoretical reference for the quality control of Periplaneta americana.

The many-banded krait, Bungarus multicinctus, has been recorded as the animal resource of JinQianBaiHuaShe in the Chinese Pharmacopoeia. Characterization of its venoms classified chief phyla of modern animal neurotoxins. However, the evolutionary origin and diversification of its neurotoxins as well as biosynthesis of its active compounds remain largely unknown due to the lack of its high-quality genome. Here, we present the 1.58 Gbp genome of B. multicinctus assembled into 18 chromosomes with contig/scaffold N50 of 7.53 Mbp/149.8 Mbp. Major bungarotoxin-coding genes were clustered within genome by family and found to be associated with ancient local duplications. The truncation of glycosylphosphatidylinositol anchor in the 3'-terminal of a LY6E paralog released modern three-finger toxins (3FTxs) from membrane tethering before the Colubroidea divergence. Subsequent expansion and mutations diversified and recruited these 3FTxs. After the cobra/krait divergence, the modern unit-B of β-bungarotoxin emerged with an extra cysteine residue. A subsequent point substitution in unit-A enabled the β-bungarotoxin covalent linkage. The B. multicinctus gene expression, chromatin topological organization, and histone modification characteristics were featured by transcriptome, proteome, chromatin conformation capture sequencing, and ChIP-seq. The results highlighted that venom production was under a sophisticated regulation. Our findings provide new insights into snake neurotoxin research, meanwhile will facilitate antivenom development, toxin-driven drug discovery and the quality control of JinQianBaiHuaShe.

Objective:To synthesize a novel site-specifically labelled probe 68Ga-1, 4, 7, 10-tetraazacyclododecane-1, 4, 7, 10-tetraacetic acid (DOTA)-Cys-Asp-Val (CDV)-Nb109 and explore its potential for detection of the programmed cell death ligand 1 (PD-L1) expression level in different tumors. Methods:Firstly, CDV was inserted into the tail of the sequence of Nb109 by genetic engineering. Then the precursor DOTA-CDV-Nb109 was prepared by mixing the maleimide-DOTA and the single-domain antibody CDV-Nb109 (amount of substance ratio 1∶1) via the maleimide-cysteine site-specific coupling strategy. Subsequently, the DOTA-CDV-Nb109 was labeled with 68Ga and purified by PD-10 column. Human melanoma A375, human PD-L1 transfected melanoma A375-hPD-L1 and human glioma U87 tumor-bearing mice models were established, and the diagnostic value of 68Ga-DOTA-CDV-Nb109 was evaluated by stability assay, cellular uptake, and microPET imaging. One-way analysis of variance and the least significant difference t test were used to analyze the data. Results:The probe 68Ga-DOTA-CDV-Nb109 was obtained with the radiochemical yield of (69.79±4.69)%, radiochemical purity more than 97%, and molar activity of (12.85±1.51) GBq/μmol. 68Ga-DOTA-CDV-Nb109 had strong binding affinity for A375-hPD-L1 with the dissociation constant ( Kd) of (66.43±17.89) nmol/L. The uptake of 68Ga-DOTA-CDV-Nb109 in A375-hPD-L1 and U87 cells were (3.17±0.15) percentage of the added radioactivity dose (%AD) and (2.08±0.03) %AD respectively, which were significantly higher than that in A375 cells ((1.21±0.14) %AD; F=82.87, t values: 15.23, 9.98, P values: <0.001, 0.003). The tumor uptake of the probe in A375-hPD-L1 ((5.21±0.35) percentage of injected dose per ml (%ID/ml)) and U87 tumor-bearing mice ((3.44±0.69) %ID/ml) were significantly higher than that in A375 tumor-bearing mice ((2.17±0.36) %ID/ml; F=249.72, t values: 35.70, 3.43, both P<0.001). Conclusion:The site-specifically labelled probe 68Ga-DOTA-CDV-Nb109, which can non-invasively and dynamically monitor the change of PD-L1 expression level in different tumors and help screen patients who can benefit from PD-L1 immune checkpoint blocking therapy, is successfully synthesized with high radiochemical purity.

Despite advances in immunotherapy for the treatment of cancers,not all patients can benefit from programmed cell death ligand 1(PD-L1)immune checkpoint blockade therapy.Anti-PD-L1 therapeutic effects reportedly correlate with the PD-L1 expression level;hence,accurate detection of PD-L1 expression can guide immunotherapy to achieve better therapeutic effects.Therefore,based on the high affinity antibody Nb109,a new site-specifically radiolabeled tracer,68Ga-NODA-cysteine,aspartic acid,and valine(CDV)-Nb109,was designed and synthesized to accurately monitor PD-L1 expression.The tracer 68Ga-NODA-CDV-Nb109 was obtained using a site-specific conjugation strategy with a radiochemical yield of about 95％and radiochemical purity of 97％.It showed high affinity for PD-L1 with a dissociation constant of 12.34±1.65 nM.Both the cell uptake assay and positron emission tomography(PET)imaging revealed higher tracer uptake in PD-L1-positive A375-hPD-L1 and U87 tumor cells than in PD-L1-negative A375 tumor cells.Meanwhile,dynamic PET imaging of a NC1-H1299 xenograft indicated that doxorubicin could upregulate PD-L1 expression,allowing timely interventional immunotherapy.In conclusion,this tracer could sensitively and dynamically monitor changes in PD-L1 expression levels in different cancers and help screen patients who can benefit from anti-PD-L1 immunotherapy.

Objective: To investigate the effect of tumor necrosis factor-alpha(TNF-α)on the immunoregulatory capacity of laryngeal mucosal mesenchymal stromal cells (LM-MSCs) and its potential molecular mechanism, and provide a theoretical basis for the study of chronic laryngitis. Methods: LM-MSCs were separated from epiglottal mucosa. The LM-MSCs cells were directly co-cultured with T cells in vitro to detect the immunomodulatory property of LM-MSCs. After long-term stimulation with inflammatory factors TNF-α in vitro, the differences were compared in the immunomodulatory ability of LM-MSCs between normal LM-MSCs and TNF-α stimulated LM-MSCs. The expression of general control non-repressed protein5(GCN5), FAS, FASL in normal LM-MSCs and TNF-α stimulated LM-MSCs was detected by Western blot and quantitative real-time RT-PCR(RT-qPCR). Results: After chronic stimulation of TNF-α, the RNA relative expression of GCN5 was 0.31±0.03 (3 days) and 0.53±0.06 (7 days) compared with control group, showing significant difference (F=13.45, P<0.05). The percentage of LM-MSC-induced T cell apoptosis was 6.27%±0.81% (3 days) and 4.99%±0.52% (7 days) in chronic stimulation group compared with control group 10.02%±1.02%. There is a significant difference among these groups (F=11.13, P<0.05). Moreover, the ability of LM-MSCs to induce T cell apoptosis is regulated by GCN5. Conclusion With the chronic stimulation of TNF-α, the expression of GCN5 in LM-MSCs is decreased, thus impairing its immunoregulatory capacity.

OBJECTIVE: To explore the genetic basis and pregnancy outcome of fetuses with urinary system anomalies. METHODS: Ultrasonographic features, genetic testing and pregnancy outcomes of 337 fetuses with urinary system anomalies identified by prenatal ultrasonograhy were collected for analysis. RESULTS: Ultrasonographic features of the fetuses were mainly characterized by hydronephrosis or hydronephrosis, polycystic kidney disease, and renal dysplasia. Thirty four fetuses (10.1%) were found to harbor a genetic defect, including 14 numerical chromosomal disorders, 10 structural chromosomal aberrations, and 10 pathogenic copy number variations (CNVs). In 31 cases, the parents elected induced labor. For the 303 fetuses with negative findings, 142 were born by spontaneous delivery or Caesarean section, 48 cases underwent induced labor, 1 case had miscarriage, and the remaining 112 cases had unknown or missed pregnancy outcomes. CONCLUSION Hydronephrosis or hydronephrosis, polycystic kidney disease, and renal dysplasia are the most common findings among fetuses with urinary system anomalies. Approximately 10.1% of such fetuses are positive by genetic testing.
Cesarean Section ; Chromosome Aberrations ; DNA Copy Number Variations ; Female ; Fetus ; Genetic Testing ; Humans ; Pregnancy ; Pregnancy Outcome ; Prenatal Diagnosis ; Ultrasonography, Prenatal

Objective To observe the safety and efficacy of preoperative Ticagrelor loading in emergency percutaneous coronary intervention (PCI)for acute ST-segment elevation myocardial infarction(STEMI).Methods A total of 213 patients with acute STEMI before undergoing emergency PCI were randomly divided into Ticagrelor group(n =105)receiving 180 mg Ticagrelor loading dose,then 90 mg twice daily and Clopidogrel group(n =108) receiving 600 mg of Clopidogrel,then 75 mg once daily.Emergency PCI postoperative coronary artery TIMI flow grade and the change of incidence of no reflow,platelet aggregation rate,incidence of bleeding events and the incidence of major adverse cardiovascular events(MACE) were compared between two groups.Results The rate of no-reflow was 7.6 ％ (8 cases)in Ticagrelor group,and 16.7 ％ (18 cases) in Clopidogrel group(x2 =3.26、P=0.030).Platelet aggregation rates at 1 h and 24 h after treatment were (55.6±4.3)％ and (48.6 ± 4.1) ％ respectively in Ticagrelor group,and (63.6 ± 3.8) ％ and (57.6 ± 3.6) ％ respectively in Clopidogrel group,which showed that platelet aggregation inhibition effect was better in Ticagrelor than in Clopidogrel (t =14.40、17.20,both P =0.001).Two groups had no major life-threatening bleeding events.Bleeding incidence had no statistically significant difference between two groups(x2 =0.14,P =0.710),and the incidence of cardiovascular adverse events showed no statistically significant difference(x2 0.04,P 0.840)between the 2 groups.Conclusions Preoperativeticagrelor loading treatment in emergency PCI therapy for acute ST segment elevation myocardial infarction shows stronger antiplatelet aggregation function,significantly improve postoperative TIMI flow,and does not increase the incidence of bleeding events.

Objective To study the effect of enhanced MK gene expression in hepatic carcinoma cells.Methods The recombinant plasmid pIRES2-EGFP-MK was transfected into SMMC 7721 cells.The mRNA and protein expression levels of MK gene in these cells were determined by real-time PCR,Western blotting and flow cytometry.The intracellular DNR accumulation of these cells was measured by flow cytometry.To investigate the effect of MK gene mediated multidrug resistance,MTT assay was employed to determine the cellular sensitivity of different chemotherapeutic drugs in MK-overexpressed SMMC 7721 cells.Results The mRNA and protein expression levels of MK gene significantly increased after the recombinant plasmid pIRES2-EGFP-MK transfected into SMMC 7721 cells,suggesting that the recombinant plasmid pIRES2-EGFP-MK can enhance the transcription of MK effectively.The DNR accumulation of MK transfected cells decreased significantly (4.06 ± 0.88,P ＜ 0.05),and IC50 of MK transfected cells to ADM/5-FU increased significantly (15 ± 3,27 ± 4,P ＜ 0.05).Conclusions After the recombinant plasmid pIRES2-EGFP-MK transfected into hepatic carcinoma cells,expression of midkine increased,enhancing the resistance of hepatic carcinoma cells to chemotherapeutic drugs.

Objective:To explore mechanisms underlying the rescuing effects of transplanted mesenchymal stem cells (MSCs) derived exosomes on estrogen-deficient osteoporosis.Methods:Mouse estrogen-deficient osteoporosis model was constructed in 12 female C57BL6/J rats and the exosome release was regulated by siRNA.Osteogenic induction,alizarin red staining and qPCR were performed to evaluate the effects of exosomes on recipient MSC functions.The miR-26a mimics and inhibitors and qPCR were used to explore the mechanisms underlying exosome-mediated functional rescue of recipient MSCs.Results:Donor MSCs alleviated estrogen-deficient osteoporosis via exosome release,and the alleviated osteoporosis by exosomes rescued the recipient MSC functions were observed.Moreover,the rescued recipient MSC functions by exosomes transferred miR-26a were found.Conclusion:Donor MSC-derived exosomes may rescue MSC functions and may remit osteoporosis of the recipient through transfering miR-26a.