Peptide-based radiopharmaceuticals targeting integrin α5β1 show promise for precise tumor diagnosis and treatment. However, current peptide-based radioligands that target α5β1 demonstrate inadequate in vivo performance owing to limited tumor retention. The use of PEGylation to enhance the tumor retention of radiopharmaceuticals by prolonging blood circulation time poses a risk of increased blood toxicity. Therefore, a PEGylation strategy that boosts tumor retention while minimizing blood circulation time is urgently needed. Here, we developed a PEGylation-enabled peptide multidisplay platform (PEGibody) for PR_b, an α5β1 targeting peptide. PEGibody generation involved PEGylation and self-assembly. [⁶⁴Cu]QM-2303 PEGibodies displayed spherical nanoparticles ranging from 100 to 200 nm in diameter. Compared with non-PEGylated radioligands, [⁶⁴Cu]QM-2303 demonstrated enhanced tumor retention time due to increased binding affinity and stability. Importantly, the biodistribution analysis confirmed rapid clearance of [⁶⁴Cu]QM-2303 from the bloodstream. Administration of a single dose of [¹⁷⁷Lu]QM-2303 led to robust antitumor efficacy. Furthermore, [⁶⁴Cu]/[¹⁷⁷Lu]QM-2303 exhibited low hematological and organ toxicity in both healthy and tumor-bearing mice. Therefore, this study presents a PEGibody-based radiotheranostic approach that enhances tumor retention time and provides long-lasting antitumor effects without prolonging blood circulation lifetime. The PEGibody-based radiopharmaceutical [⁶⁴Cu]/[¹⁷⁷Lu]QM-2303 shows great potential for positron emission tomography imaging-guided targeted radionuclide therapy for α5β1-overexpressing tumors.

The activation proteins released by fibroblasts in the tumor microenvironment regulate tumor growth, migration, and treatment response, thereby influencing tumor progression and therapeutic outcomes. Owing to the proliferation and metastasis of tumors, fibroblast activation protein (FAP) is typically highly expressed in the tumor stroma, whereas it is nearly absent in adult normal tissues and benign lesions, making it an attractive target for precision medicine. Radiolabeled agents targeting FAP have the potential for targeted cancer diagnosis and therapy. This comprehensive review aims to describe the evolution of FAPI-based radiopharmaceuticals and their structural optimization. Within its scope, this review summarizes the advances in the use of radiolabeled small molecule inhibitors for tumor imaging and therapy as well as the modification strategies for FAPIs, combined with insights from structure-activity relationships and clinical studies, providing a valuable perspective for radiopharmaceutical clinical development and application.

Sequencing-based spatial transcriptomics(ST)is an emerging technology to study in situ gene expression patterns at the whole-genome scale.Currently,ST data analysis is still complicated by high technical noises and low resolution.In addition to the transcriptomic data,matched histopathological images are usually generated for the same tissue sample along the ST experiment.The matched high-resolution histopathological images provide complementary cellular phenotypi-cal information,providing an opportunity to mitigate the noises in ST data.We present a novel ST data analysis method called transcriptome and histopathological image integrative analysis for ST(TIST),which enables the identification of spatial clusters(SCs)and the enhancement of spatial gene expression patterns by integrative analysis of matched transcriptomic data and images.TIST devises a histopathological feature extraction method based on Markov random field(MRF)to learn the cellular features from histopathological images,and integrates them with the transcrip-tomic data and location information as a network,termed TIST-net.Based on TIST-net,SCs are identified by a random walk-based strategy,and gene expression patterns are enhanced by neighborhood smoothing.We benchmark TIST on both simulated datasets and 32 real samples against several state-of-the-art methods.Results show that TIST is robust to technical noises on multiple analysis tasks for sequencing-based ST data and can find interesting microstructures in dif-ferent biological scenarios.TIST is available at http://lifeome.net/software/tist/and https://ngdc.cncb.ac.cn/biocode/tools/BT007317.

Objective:To explore the protective effects of bradykinin postconditioning on cardiopulmonary resuscitation (CPR) rats, and to assess the underlying mechanisms.Methods:Forty-eight adult male Sprague-Dawley (SD) rats were randomly divided into four groups according to random number table: Sham operation group, cardiac arrest (CA) group, bradykinin treatment (BK) group, and AMP-activated protein kinase (AMPK) inhibitor Compound C+ bradykinin treatment (CP+BK) group, finally, 8 rats in each group were taken for follow-up experiment. CA was induced by asphyxia. Rats in the Sham group received arteriovenous catheterization, endotracheal intubation, and mechanical ventilation, without CA. Compound C (250 μg/kg) was intraperitoneally injected in CP+BK group 30 minutes before CA, and the same volume of dimethyl sulfoxide (DMSO) was given in the remaining groups. Bradykinin (150 μg/kg) was intraperitoneally injected in BK group and CP+BK group 48 hours after restoration of spontaneous circulation (ROSC), and same volume of saline was given in the remaining groups. The neural function of rats in each group was evaluated with neurological deficit score (NDS) 72 hours after ROSC. Microtubule-associated protein light chain 3 (LC3) and p62 expressions were detected by immunohistochemistry, autophagosomes were observed by transmission electron microscopy, and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling method (TUNEL) assay was used to assess apoptosis.Results:Compared with the Sham group, the NDS was decreased (60.75±5.80 vs. 80.00±0.00, P < 0.01), the expression levels of LC3 and p62 elevated [LC3 ( A value): 1.04±0.64 vs. 0.40±0.14, p62 ( A value): 2.75±0.57 vs. 0.36±0.12, both P < 0.05], the number of autophagosomes and apoptotic cells increased in the CA group [(39.00±8.00)% vs. (3.87±1.90)%, P < 0.05]. Compared with the CA group, the NDS (67.75±6.32 vs. 60.75±5.80, P < 0.05), the expression of LC3 ( A value: 1.60±0.34 vs. 1.04±0.64, P < 0.05), and the number of autophagosomes increased in the BK group, while the expression of p62 and the rate of apoptotic cells reduced [p62 ( A value): 1.51±0.32 vs. 2.75±0.57, apoptotic cells rate: (23.03±1.91)% vs. (39.00±8.00)%, both P < 0.05]. Compared with the BK group, the NDS (59.00±8.19 vs. 67.75±6.32, P < 0.05), the expression of LC3 ( A value: 0.62±0.41 vs. 1.60±0.34, P < 0.05) and the number of autophagosomes declined in the CP+BK group, while the expression of p62 and the rate of apoptotic cells elevated [p62 ( A value): 3.50±0.47 vs. 1.51±0.32, apoptotic cells rate: (44.53±10.15)% vs. (23.03±1.91)%, both P < 0.05]. Conclusion:Bradykinin postconditioning played a neuroprotective role in CPR rats by activating autophagy and reducing apoptosis.

PLCζ is a new isoenzyme of the PLC family which plays an important role in activating mammalian oocytes. In recent years, large-scale expression and purification of active PLCζ protein in vitro for structural biology research has not been successful. In this study, the recombinant human PLCζ protein was expressed and purified in the baculovirus expression system. First, the full length of human PLCζ gene was cloned into the pFastBac-HTA plasmid to form the recombinant donor plasmid that was further transformed into DH10Bac Escherichia coli cells to construct the recombined bacmid by the site-specific transposition that was screened by resistance and blue-white spots. Then the bacmid was transfected to Sf9 insect cells via cellfectin to package the recombinant baculovirus. After the amplification of the recombinant baculovirous, the recombinant protein was expressed from the cells transduced by the recombinant baculovirus and was purified by Ni-NTA resin. Purified protein was identified by Western blotting and time-of-flight mass spectrometry and the enzyme activity was determined. The results showed that the recombinant PLCζ protein in the Sf9 cells was achieved at 72 hours after baculovirus infection and expressed in secreted form in cell culture medium. The recombinant protein purified by Ni²⁺ affinity column was identified as PLCζ by Western blotting and ionization time-of-flight mass spectrometry and the enzyme activity was up to 326.8 U/mL. The experimental results provide a reference for the large-scale production and biological application of recombinant human PLCζ protein.
Animals ; Baculoviridae ; Genetic Vectors ; Humans ; Recombinant Proteins ; Sf9 Cells ; Spodoptera

Objective To investigate the application value of dynamic single-photon emission computed tomography (SPECT) 99m-technetium galactosyl human serum albumin diethylenetriamine pentaacetic acid injection (99 Tcm-GSA) scintigraphy assessing regional liver function changes before and after portal vein embolization (PVE).Methods The retrospective cross-sectional study was conducted.The clinical data of 11 patients with Bismuth Ⅲ a hilar cholangiocarcinoma who were admitted to the General Hospital of People's Liberation Army (10 patients) and Beijing Tsinghua Changgung Hospital (1 patient) from October 2010 to October 2016 were collected.B ultrasound-guided percutaneous transhepatic ipsilateral exbolization was performed before radical resection of hilar cholangiocarcinoma.Dynamic SPECT 99 Tcm-GSA scintigraphy was performed to calculate and compare the changes of functional liver volume (FLV),morphological liver volume (MLV) and functional liver density (FLD) in embolized lobe and non-embolized lobe before PVE and 2 weeks after PVE.Observation indicators:(1) the changes of serum indexes in 2 weeks before and after PVE;(2) the changes of FLV,MLV and FLD in the whole liver,embolized and non-embolized lobes in 2 weeks before and after PVE;(3) surgical and postoperative situations of hilar cholangiocarcinoma;(4) follow-up and survival situations.Follow-up using outpatient examination and telephone interview was performed to detect postoperative serum toal bilirubin (TBil) level,with or without peritoneal effusion and survival up to June 2017.Measurement data with normal distribution were represented as x-±s.The comparisons of pre-and post-operative data were analyzed by the paired t test.Results (1) The changes of serum indexes in 2 weeks before and after PVE:11 patients underwent successful right PVE.The alanine aminotransferase (ALT),TBil,albumin (Alb),Platelets (PLT) and prothrombin time (PT) were respectively (113±20) U/L,(73± 8) μmol/L,(35.0±2.5) g/L,(209±58) × 109/L,(11.4±0.7) seconds in 2 weeks before PVE and (120± 18) U/L,(36± 7) μmol/L,(34.4± 3.2) g/L,(224± 82) × 109/L,(11.2±0.8)seconds in 2 weeks after PVE,with a statistically significant difference in TBil level (t=-10.592,P＜0.05) and no statistically significant difference in ALT,Alb,PLT and PT (t=0.981,-0.350,-0.591,0.533,P＞0.05).(2) The changes of FLV,M LV and FLD in the whole liver,embolized and nonembolized lobes in 2 weeks before and after PVE:the FLV,MLV and FLD of the whole liver were respectively (894±255) mL,(1 552±504) mL,0.59±0.14 in 2 weeks before PVE and (812±206) mL,(1 521±422) mL,0.55±0.16 in 2 weeks after PVE,with no statistically significant difference (t =1.569,0.666,1.980,P＞ 0.05).The FLV,MLV and FLD of the embolized lobe were respectively (623±275) mL,(1 047± 394) mL,0.62±0.14 in 2 weeks before PVE and (375±240) mL,(865±337) mL,0.44±0.24 in 2 weeks after PVE,with statistically significant differences (t =5.909,3.736,3.359,P ＜ 0.05);the descending percentages were respectively 38.1％,9.8％ and 24.6％.The FLV,MLV and FLD of the non-embolized lobe were respectively (274±152)mL,(530±176)mL,0.52±0.21 in 2 weeks before PVE and (436±149) mL,(656±133)mL,0.68± 0.24 in 2 weeks after PVE,with statistically significant differences (t =-6.019,-6.345,-3.933,P＜0.05);the elevated percentages were respectively 80.1％,19.9％ and 23.8％.(3) Surgical and postoperative situations of hilar cholangiocarcinoma:of 11 patients,10 received successful peri-hilar right hemihepatectomy,the right hepatic atrophy and an obvious demarcation line between left and right liver were found intraoperatively;1 stopped operation due to detect intraoperatively peritoneal metastasis of tumor.The operation time,volume of intraoperative blood loss and time of postoperative abdominal drainage-tube removal were respectively (585± 194)minutes,(472± 274)mL and (8±5)days.Of 10 patients undergoing operations,2 were complicated with massive peritoneal effusion at 2 days postoperatively,volume of peritoneal effusion remained more than 500 mL up to 7 days after drainage,and were improved by 1-month conservative treatment;other 8 patients were not complicated with hepatic dysfunction.Duration of hospital stay of 11 patients was (16± 4) days.(4) Follow-up and survival situations:10 patients were followed up for 4-72 months,with a median time of 39 months.During the follow-up,there was no evaluated TBil level and peritoneal effusion in 10 patients.The median survival time,1-,3-and 5-year overall survival rates were 88.8％,74.6％ and 36.8％,respectively.Conclusions The dynamic SPECT 99Tcm-GSA scintigraphy can effectively evaluate liver function changes of embolized and non-embolized lobes before and after PVE.The increased rate of FLV of non-embolized lobe is higher than that of MLV.

Objective To investigate the changes of diffusion tensor imaging (DTI) in the cerebral white matter of rats with ischemia-reperfusion injury.Methods Adult SD rats were randomly divided into two groups,sham operation group and model group.The model of ischemia reperfusion injury was made by the Koizumi suture method to occlude the middle cerebral artery.Application of Zea-Longa score was carried out to determine the establishment of modeling,and the modified neurological severity score (mNSS) was used to evaluate the neurological deficit of rats.The Rotarod test instrument was used to observe the motor function of rats by using Rotarod fatigue balance signs,and the DTI sequence scanning observation of brain white matter nerve fiber damage was determined by using Brook 7.0T small animal magnetic resonance imaging system.Track Vis software was used to analyze the distribution of cerebral white matter nerve fibers,and the relative number of nerve fibers in the areas of ROI (region of interest,ROI),sensorimotor areas and striatum were calculated.Results The results showed varying degrees of neurological impairment in rats 2 h after cerebral ischemia reperfusion injury,and the Zea-Longa score and the mNSS score were gradually reduced at the 1 d,7 d and 14 d after ischemia reperfusion injury.The time of rats retaining on the rotating rod was shortened at the 7 d and 14 d after ischemia reperfusion injury.At the ischemic lateral,nerve fibers decreased significantly,and the number of sensory nerve fiber connections in the sensorimotor areas to striatum was reduced.Nissl staining showed that the cytoplasm of neurons in the sensorimotor cortex and striatum of the ischemic lateral were disappeared and the Nissl bodies were decreased.Conclusions The nerve fibers of sensory motor cortex connecting to striatum were damaged by ischemia reperfusion injury.

Objective To investigate the molecular mechanism of the effect of CYP2C19 polymorphism on the efficacy of clopidogrel in patients with stable coronary artery disease (SCAD).Methods A total of 208 patients with coronary artery disease undergoing percutaneous coronary intervention (PCI) were included.The CYP2C19 variant alleles were detected by gene sequencing.Platelet reactivity was assessed by thrombelastograph at 24 h after 300 mg clopidogrel loading.P2Y12-Gi signaling was measured by flow cytometric analysis of vasodilator-stimulated phosphorylation-platelet reactivity index (VASP-PRI) and Akt phosphorylation (P-Akt) index.Results Among 208 patients,40.9％ were classified as CYP2C19 * 1/* 1 (non-carriers) with no loss-of-function (LOF),44.7％ as CYP2C19 * 1/* 2 or CYP2C19 * 1/* 3 (one LOF carriers) and 14.4％ as CYP2C19 * 2/*2 or CYP2C19 * 2/* 3 (two LOF carriers).Both postclopidogrel platelet aggregation and VASP-PRI increased by the presence of one LOF allele,and were even more pronounced with the presence of two LOF alleles.In contrast,P-Akt index only increased in two LOF carriers.VASP-PRI was positively associated with postclopidogrel platelet aggregation (r =0.661,P＜0.001),but not with P-Akt index.Conclusions The two CYP2C19 LOF carriage was associated with more active state of P2Y12-Gi signaling in postclopidogrel platelet in Chinese patients with SCAD.

UNLABELLEDObjective To explore the effects of sperm DNA integrity rate, acrosome integrity rate and acrosome reaction rate on the outcomes of rescue intracytoplasmic sperm injection (ICSI).

METHODSThis retrospective analysis was conducted among 97 infertile couples receiving rescue ICSI due to failure of in vitro fertilization procedures in our Reproductive Medicine Center. Of these 97 women, 41 had clinical pregnancy and 56 did not, and the effects of sperm DNA integrity rate (estimated by DNA fragmentation index, DFI), acrosome integrity rate and acrosome reaction rate on rescue ICSI outcomes were analyzed.

RESULTSNo significant difference was found in paternal age, testosterone value, testicular volume, FSH, female patient' age or the number of eggs retrieved between the two groups (P>0.05), but the infertility years was significantly shorter in the pregnancy group than in the non-pregnancy group (P<0.05). The fertilization rate and cleavage rate were similar between the two groups (P>0.05), but the good embryo rate was significantly higher in the pregnancy group (P<0.05). The sperm DNA integrity or acrosome reaction rate did not differ significantly between the two groups (P>0.05), but the acrosome integrity rate was significantly higher in the pregnancy group (P<0.05). The sperm DNA integrity rate, acrosome integrity or acrosome reaction rate were not correlated with the fertilization rate, cleavage rate or good embryo rate (P>0.05). The pregnancy rate, twin and single fetus rates were 42.3%, 10.3% and 32.0% in this cohort after recue ICSI, respectively.

CONCLUSIONRescue ICSI is an effective treatment after failed in vitro fertilization procedure, and sperm acrosome integrity rate is associated with the outcome of rescue ICSI.

Acrosome ; pathology ; Acrosome Reaction ; DNA Fragmentation ; Female ; Fertilization ; Fertilization in Vitro ; Humans ; Infertility ; Male ; Pregnancy ; Pregnancy Rate ; Retrospective Studies ; Sperm Injections, Intracytoplasmic

Objective: To develop a high efficient expression, purification system of recombinant arginine deiminase(ADI).Methods: cDNA fragment encoding for mycoplasma ADI was obtained by artificial synthesis and was cloned into prokaryotic expression vector(pBV220). The recombinant ADI was generated by the transformation of the recombinant vector into the host strain DH5?. Anion exchange and gel filtration chromatography was carried out for purification of the recombinant ADI. The biological activity of final product was detected by the assay of agrinine degradation in vitro. Results: A prokaryotic expression plasmid pBV220-ADI was generated successfully, and was identified by DNA sequencing; the recombinant protein was highly expressed in DH5?, the proportion of the recombinant protein is exceeded 35% of the whole protein. The inclusion bodies were solubilized with 6mol/L guanidine hydrochloride under reducing conditions in order to avoid incorrect disulfide-bond formation of the recombinant ADI molecules. Dilution and dialysis at lower degrees temperature were the optimum renaturation methods. After gel filtration, the purity and specific activity of rADI reached 95% and 80 IU/mg respectively. Conclusions: A set of protocols for high efficient rADI expression and purification has been established, which is simple, efficient and applicable.