Mitochondria-specific near-infrared photoactivation of peroxynitrite upconversion luminescent nanogenerator for precision cancer gas therapy

Gas therapy is emerging as a highly promising therapeutic strategy for cancer treatment. However, there are limitations, including the lack of targeted subcellular organelle accuracy and spatiotemporal release precision, associated with gas therapy. In this study, we developed a series of photoactivatable nitric oxide (NO) donors NRh-R-NO (R = Me, Et, Bn, iPr, and Ph) based on an N-nitrosated upconversion luminescent rhodamine scaffold. Under the irradiation of 808 nm light, only NRh-Ph-NO could effectively release NO and NRh-Ph with a significant turn-on frequency upconversion luminescence (FUCL) signal at 740 nm, ascribed to lower N-N bond dissociation energy. We also investigated the involved multistage near-infrared-controlled cascade release of gas therapy, including the NO released from NRh-Ph-NO along with one NRh-Ph molecule generation, the superoxide anion O2⋅- produced by the photodynamic therapy (PDT) effect of NRh-Ph, and highly toxic peroxynitrite anion (ONOO‒) generated from the co-existence of NO and O2⋅-. After mild nano-modification, the nanogenerator (NRh-Ph-NO NPs) empowered with superior biocompatibility could target mitochondria. Under an 808 nm laser irradiation, NRh-Ph-NO NPs could induce NO/ROS to generate RNS, causing a decrease in the mitochondrial membrane potential and initiating apoptosis by caspase-3 activation, which further induced tumor immunogenic cell death (ICD). In vivo therapeutic results of NRh-Ph-NO NPs showed augmented RNS-potentiated gas therapy, demonstrating excellent biocompatibility and effective tumor inhibition guided by real-time FUCL imaging. Collectively, this versatile strategy defines the targeted RNS-mediated cancer therapy.

Application progress on in vivo drug analysis technique in clinical pharmacy / 药学实践杂志

Objective To explore the progress on the application of in vivo drug analysis techniques in clinical pharmacy work. Methods Relevant literature was reviewed to provide an overview of the characteristics of clinical samples, common in vivo drug analysis methods used in the clinic, the application and existing problems of in vivo drug analysis in clinical pharmacy. Results and Conclusion In recent years, with the increasing demand for individualized and precise treatment in clinical practice and the continuous development of analytical techniques, in vivo drug analysis techniques have been widely used in clinical pharmacy work, which have become one of the important auxiliary techniques to promote rational clinical drug use, improve individualized treatment and reduce the occurrence of adverse reactions. However, in the actual application, there were still problems such as the invasive blood sampling that hinders sampling, the weak ability to interpret drug monitoring results and clinical testing methods that still need to be improved. These problems should be taken seriously and continuously improved and solved in the subsequent research and application.

Preparation and in vivo Distribution of Essential Oil from Alpinia zerumbet Fructus Encapsulated Nanoemulsions / 中国实验方剂学杂志

ObjectiveTo prepare oral nanoemulsions encapsulating essential oil from Alpinia zerumbet fructus（EOFAZ） and to investigate its pro-absorption effect in vitro and distribution in vivo. MethodThe proteoglycan conjugate polysaccharides of vinegar-processed Bupleuri Radix-bovine serum albumin（VBCP-BSA） was prepared by Maillard reaction of VBCP and BSA. Taking VBCP-BSA as emulsifier， vitamin B12（VB12） as absorption enhancer， and medium chain triglycerides mixed with EOFAZ as oil phase， the nanoemulsions loaded with EOFAZ was prepared by high energy emulsification method. The particle size， particle size distribution， surface Zeta potential， EOFAZ content and appearance and morphology of the nanoemulsions were characterized， and fluorescein tracer method was used to investigate the absorption effect of fluorescein-labeled EOFAZ nanoemulsions in vitro and their distribution in vivo. ResultVBCP-BSA was formed by Maillard reaction for 48 h with high grafting rate. Using VBCP-BSA as emulsifier， the homogeneous pink nanoemulsions was prepared and denoted as EOFAZ@VBCP-BSA/VB12. The particle size of the nanoemulsions was less than 100 nm and the particle size distribution was uniform. The surface of the nanoemulsions was a weak negative charge， and the shape was spherical. The encapsulation rate of the nanoemulsions for EOFAZ was greater than 80%， which had a good absorption effect in vitro and could enhance liver accumulation after oral administration. ConclusionThe designed proteoglycan nanoemulsions can effectively load EOFAZ， promote oral absorption and enhance liver distribution， which can provide experimental basis for the development of oral EOFAZ liver protection preparations.

Analysis on Components Absorbed into Blood and Cerebrospinal Fluid of Schisandrae Chinensis Fructus Based on Sequential Metabolism / 中国实验方剂学杂志

ObjectiveTo identify the prototypical components and metabolites absorbed into blood and cerebrospinal fluid of Schisandrae Chinensis Fructus（SCF） based on sequential metabolism combined with liquid chromatography-mass spectrometry. MethodBlood and cerebrospinal fluid samples of integrated metabolism， intestinal metabolism and hepatic metabolism were collected from male SD rats after gavage and in situ intestinal perfusion administration， and ultra-performance liquid chromatography-quadrupole/electrostatic field orbitrap high-resolution mass spectrometry（UPLC Q-Exactive Orbitrap MS） was used to analyze and compare the differences in the spectra of SCF extract， blank plasma， administered plasma， blank cerebrospinal fluid and administered cerebrospinal fluid with ACQUITY UPLC BEH Shield RP18 column（2.1 mm×100 mm， 1.7 µm）， the mobile phase was acetonitrile（A）-0.1% formic acid aqueous solution（B） for gradient elution（0-7 min， 95%B； 7-12 min， 95%-35%B； 12-17 min， 35%-15%B； 17-20 min， 15%-12%B； 20-22 min， 12%-5%B； 22-23 min， 5%B； 23-25 min， 5%-95%B； 25-28 min， 95%B）. And heated electrospray ionization（HESI） was used with positive and negative ion modes， the scanning range was m/z 100-1 500. The prototypical constituents and their metabolites absorbed into blood and cerebrospinal fluid of SCF were identified according to the retention time， characteristic fragments， molecular formulae and the information of reference substances. ResultA total of 42 chemical components were identified in the extract of SCF， including lignans， flavonoids， amino acids， tannins， and others， of which lignans were the main ones. A total of 27 prototypical components and 14 metabolites were identified in plasma samples from different sites. A total of 15 prototypical components and 9 metabolites were identified in cerebrospinal fluid. The main metabolic reactions involved in the formation of metabolites were mainly demethylation， methylation， demethoxylation and hydroxylation. ConclusionThrough the systematic identification of the prototypical components and metabolites of SCF in rats， it provides data support for further better exploring the material basis of SCF in the treatment of central nervous system diseases.

Genotoxic and cytotoxic evaluation of venlafaxine in an acute and a subchronic assay in mouse

Abstract The present research was made to determine the micronuclei and cytotoxic capacity of the antidepressant venlafaxine in an in vivo acute and subchronic assays in mouse. In the first study, we administered once 5, 50, and 250 mg/kg of the drug, and included a negative and a daunorubicin treated group. Observations were daily made during four days. The subchronic assay lasted 5 weeks with daily administration of venlafaxine (1, 5, and 10 mg/kg) plus a negative and an imipramine administered groups. Observations were made each week. In the first assay results showed no micronucleated polychromatic erythrocytes (MNPE) increase, except with the high dose at 72 h. The strongest cytotoxic effect was found with 250 mg/kg at 72 h (a 51% cytotoxic effect in comparison with the mean control level). In the subchronic assay no MNPE increase was found; however, with the highest dose a significant increase of micronucleated normochromatic erythrocytes was observed in the last three weeks (a mean of 51% respect to the mean control value). A cytotoxic effect with the two high doses in the last two weeks was observed (a polychromatic erythrocyte mean decrease of 52% respect to the mean control value). Results suggest caution with venlafaxine.

Resumo A presente pesquisa foi feita para determinar a capacidade micronuclei e citotóxica do antidepressivo venlafaxina em ensaios agudos e subcrônicos in vivo em camundongos. No primeiro estudo, administramos uma vez 5, 50 e 250 mg/kg do medicamento e incluímos um grupo negativo e um grupo tratado com daunorubicina. As observações foram feitas diariamente durante quatro dias. O ensaio subcrônico durou cinco semanas com administração diária de venlafaxina (1, 5, e 10 mg/kg) mais um grupo negativo e um grupo administrado de imipramina. As observações foram feitas a cada semana. No primeiro ensaio, os resultados não mostraram aumento de eritrócitos policromáticos micronucleados (MNPE), exceto com a dose elevada a 72 h. O efeito citotóxico mais forte foi encontrado com 250 mg/kg a 72 h (um efeito citotóxico de 51% em comparação com o nível médio de controle). No ensaio subcrônico não foi encontrado aumento de MNPE; entretanto, com a dose mais alta, um aumento significativo de eritrócitos normocromáticos micronucleados foi observado nas últimas três semanas (média de 51% em relação ao valor médio de controle). Foi observado um efeito citotóxico com as duas altas doses nas últimas duas semanas (uma diminuição média de 52% em relação ao valor médio de controle dos eritrócitos policromáticos). Os resultados sugerem cautela com a venlafaxina.

Genotoxic and cytotoxic evaluation of venlafaxine in an acute and a subchronic assay in mouse / Avaliação genotóxica e citotóxica da venlafaxina em ensaio agudo e subcrônico em camundongos

Abstract The present research was made to determine the micronuclei and cytotoxic capacity of the antidepressant venlafaxine in an in vivo acute and subchronic assays in mouse. In the first study, we administered once 5, 50, and 250 mg/kg of the drug, and included a negative and a daunorubicin treated group. Observations were daily made during four days. The subchronic assay lasted 5 weeks with daily administration of venlafaxine (1, 5, and 10 mg/kg) plus a negative and an imipramine administered groups. Observations were made each week. In the first assay results showed no micronucleated polychromatic erythrocytes (MNPE) increase, except with the high dose at 72 h. The strongest cytotoxic effect was found with 250 mg/kg at 72 h (a 51% cytotoxic effect in comparison with the mean control level). In the subchronic assay no MNPE increase was found; however, with the highest dose a significant increase of micronucleated normochromatic erythrocytes was observed in the last three weeks (a mean of 51% respect to the mean control value). A cytotoxic effect with the two high doses in the last two weeks was observed (a polychromatic erythrocyte mean decrease of 52% respect to the mean control value). Results suggest caution with venlafaxine.

Resumo A presente pesquisa foi feita para determinar a capacidade micronuclei e citotóxica do antidepressivo venlafaxina em ensaios agudos e subcrônicos in vivo em camundongos. No primeiro estudo, administramos uma vez 5, 50 e 250 mg/kg do medicamento e incluímos um grupo negativo e um grupo tratado com daunorubicina. As observações foram feitas diariamente durante quatro dias. O ensaio subcrônico durou cinco semanas com administração diária de venlafaxina (1, 5, e 10 mg/kg) mais um grupo negativo e um grupo administrado de imipramina. As observações foram feitas a cada semana. No primeiro ensaio, os resultados não mostraram aumento de eritrócitos policromáticos micronucleados (MNPE), exceto com a dose elevada a 72 h. O efeito citotóxico mais forte foi encontrado com 250 mg/kg a 72 h (um efeito citotóxico de 51% em comparação com o nível médio de controle). No ensaio subcrônico não foi encontrado aumento de MNPE; entretanto, com a dose mais alta, um aumento significativo de eritrócitos normocromáticos micronucleados foi observado nas últimas três semanas (média de 51% em relação ao valor médio de controle). Foi observado um efeito citotóxico com as duas altas doses nas últimas duas semanas (uma diminuição média de 52% em relação ao valor médio de controle dos eritrócitos policromáticos). Os resultados sugerem cautela com a venlafaxina.

Small-fibre neuropathy in leprosy the role of in vivo confocal microscopy: A cross-sectional study

Background: Leprosy (or Hansen’s disease) continues to present considerable challenges regarding containment and early diagnosis. Leprosy is considered to be primarily a neural disease that first affects the sensory function of small fibres. Although the condition is well described in terms of clinical manifestations and histology, few studies have been undertaken to detect damage done to small-fibre sensory nerves. In vivo confocal microscopy is a useful tool for conducting a detailed evaluation of these structures, although its use in individuals affected by leprosy has still not been explored. Objective: To evaluate in vivo confocal microscopy findings in Hansen’s disease patients and their association with clinical variables relating to this disease. Method: A cross-sectional case-series type study was carried out between October 2019 and May 2021, in Recife, Pernambuco, Brazil. Socio-demographic and clinical data were gathered from 21 patients with leprosy. The douleur neuropathique 4 neuropathic pain questionnaire was used to evaluate pain. In vivo confocal microscopy of the cornea was employed to evaluate the small-calibre fibres. Findings were compared with those for a control group of 23 healthy individuals. Results: In relation to clinical parameters, 90.5% of the patients were classified as “multibacillary” according to the World Health Organization criteria, and 70% as dimorphic or borderline, in accordance with the Madrid classification. Around 52.4% had received a diagnosis after one year or less of living with the disease, while 95.2% presented alterations in small-fibre sensory function and 35% presented such alterations in the large fibre. Neuropathic pain was present in 81% of the patients. In vivo confocal microscopy found no statistically significant difference in mean age and distribution according to sex between the Hansen disease patients and the control group of healthy individuals. The median-of-means for dendritic cells and volume of sub-basal nerve fibres in the control group were used to test for normality. Both eyes of all leprosy patients examined contained higher number of dendritic cells than the median value and a volume of sub-basal nerve fibres lower than the mean. These differences were statistically significant (P < 0.001 and P < 0.001, respectively). Multibacillary individuals had a median number of dendritic cells two times that of paucibacillary individuals (P = 0.035). Limitations: No association was found between the variables examined using in vivo confocal microscopy and clinical variables relating to small-fibre damage, the neuropathic pain questionnaire or alterations detected by the neurological examination. We believe, however, that Cochet-Bonnet esthesiometry of the cornea may have revealed such an association. Conclusion: In vivo confocal microscopy is a useful diagnostic tool for detecting small fibre loss in individuals affected by leprosy and may constitute a useful addition to the range of tools available to help curb the effects of neuropathy in these patients.

Internal occupational dosimetry for the occupational worker exposed to [18F] FDG from the Cyclotron-PET/CT Laboratory of the Universidad de Costa Rica / Dosimetría ocupacional interna para el personal trabajador expuesto a [18F] FDG del ciclotrón-PET/CT en el Laboratorio de la Universidad de Costa Rica

ABSTRACT: The aim of this work is to provide a methodology for evaluating the committed effective dose E(50) due to the incorporation of [18F] FDG in the occupationally exposed worker (OEW) of the Cyclotron-PET/CT Laboratory of the Centro de Investigación en Ciencias Atómicas, Nucleares y Moleculares (CICANUM) at Universidad de Costa Rica using in vivo measurements. The measurement system was calibrated to perform in vivo measurements and defined as the corresponding bioassay function for the radiopharmaceutical used. The conversion factor was assessed with a known activity of 18F in the geometry and measurement time established. Among the most relevant results, the measurement parameters and the calibration procedure were defined. A value of 1.73 x 103 Bq/cps for in vivo brain measurements was obtained as a conversion factor. This study provides a methodology, to evaluate the committed effective dose due to the incorporation of 18F-FDG in a radionuclide production and diagnostic center

Advances in approved nucleic acid drugs and lipid nanoparticle system / 药学学报

Nucleic acids, as a next generation of biotechnology drugs, not only can fundamentally treat diseases, but also own significant platform characteristics in view of technology and production. Therefore, nucleic acid-based drugs have broad clinical applications in biomedical fields. However, nucleic acids are degradable and unstable, and have very low intracellular delivery efficiency in vitro and in vivo, which greatly limits their applications. In recent years, ionizable lipid-based lipid nanoparticles have shown promising application potentials and have been successfully applied to COVID-19 (Coronavirus Disease 2019) vaccines in clinic. Lipid nanoparticles demonstrate high in vivo delivery efficiency and good safety profile due to their unique structural and physicochemical properties, which provides many possibilities for their clinical applications for nucleic acid delivery in the future. This review focused on the characteristics of nucleic acid drugs and their delivery barriers, and discussed the approved nucleic acid drugs to illustrate the key aspects of the success of their delivery carrier system. In addition, problems to be solved in the field were highlighted.

Study on long-acting analgesic lappaconitine hydrobromide lyotropic liquid crystal injection / 药学学报

Long-acting analgesia is a common clinical treatment method after surgery. The slow-release injection with long-acting analgesia has the advantages of less medication frequency and stable effect. In this study, the analgesic drug lappaconitine hydrobromide lyotropic liquid crystal injection was prepared, and its sustained release mechanism, drug release and pharmacodynamic characteristics were evaluated. The results of polarizing microscope and freeze-transmission electron microscope showed that the lyotropic liquid crystal injection of the liquid crystal precursor preparation of lappaconitine hydrobromide could be obtained by the combination of glycerol monooleate (GMO) and soybean lecithin (SPC) in different proportions. The results of dissolution study in vitro showed that the drug release rate of different forms of liquid crystal preparations was layered liquid crystal > cubic liquid crystal > hexagonal liquid crystal. The mathematical model fitting results of the release data showed that the external release of layered liquid crystal, cubic liquid crystal and hexagonal liquid crystal conforms to the Ritger-Peppas model, and the release mechanism was Fick diffusion. The results of pharmacodynamics study in vivo showed that the analgesic effect of lappaconitine hydrobromide lyotropic liquid crystal injection lasted for 3 days, and there was no abnormality in the incision and local tissue, showing good safety and tolerance. The study on drug release and elimination process of the in vivo gel repository showed that lappaconitine hydrobromide could be completely released from the lyotropic liquid crystal 3 days after administration, and the sustained-release materials could be gradually eliminated locally. All animal experiments were approved by the Experimental Animal Ethics Committee of the Shanghai Institute of Materia Medica, Chinese Academy of Sciences (No. 2021-08-GY-61) and the experiments were conducted in accordance with the relevant guiding principles and regulations. The lyotropic liquid crystal injection of lappaconitine hydrobromide prepared in this study presented a solution state at room temperature, and underwent phase transition in contact with the body fluid at the administration site, formed a drug depot and exerted a slow drug release effect. This preparation can reduce systemic toxicity, prolong the duration of analgesia, reduce the number of administrations, improve the compliance of postoperative patients, and provide a reference for the design of long-term sustained release analgesic preparations.

Study on preparation and in vivo safety of progesterone cocrystal / 中国药房

OBJECTIVE To prepare progesterone-2-chloro-4-nitroaniline cocrystal （CNA） so as to improve the solubility of progesterone and primarily evaluate the safety of the progesterone cocrystal in vivo. METHODS Using progesterone as the main body and CNA as the ligand， progesterone-CNA cocrystal was prepared with solvent evaporation method. The cocrystal was characterized by X-ray single crystal diffraction， X-ray powder diffraction （XRPD）， differential scanning calorimetry （DSC） and Fourier transform infrared spectroscopy （IR）. The dissolution rate of cocrystal was compared with those of progesterone and physical mixture. Forty-eight female KM mice were randomly divided into normal group （phosphate buffer containing 0.1% dimethyl sulfoxide）， progesterone group （16 mg/kg）， CNA group （9 mg/kg）， progesterone-CNA cocrystal low-dose， medium- dose and high-dose groups （6， 12.5， 25 mg/kg）， with 8 mice in each group. They were given relevant medicine/solvent intramuscularly， once a day， for consecutive 14 d. The safety of cocrystal was evaluated primarily by determining/observing the changes in body weight， organ index， tissue morphology， blood routine indicators， and liver and kidney function indicators. RESULTS The new crystal structure in the X-ray single crystal diffraction results， the new characteristic peak in the XRPD pattern， the change of melting point in the DSC results， and the change of the characteristic peak position in the range of 3 500- 2 750 cm－1 and 1 700-1 250 cm－1 in the infrared spectrum all Δ 基金项目国家重点研发计划项目（No.2022YFC3502100） indicated that progesterone-CNA cocrystal was successfully ＊第一作者 硕士研究生 。研究方向 ：药物制备技术与工艺 。 prepared， and the dissolution rate of cocrystal was more than E-mail：SWB_1221@163.com # 通信作者教授，硕士生导师，博士。研究方向：药物制备技术与 twice that of the progesterone raw material drug. The results of 工艺。E-mail：wuxx-415@126.com in vivo safety experiments showed that the mortality rate of all 中国药房 2023年第34卷第13期 China Pharmacy 2023 Vol. 34 No. 13 · 1567 · groups was zero. Compared with normal group， uterine indexes of mice in progesterone group and progesterone-CNA cocrystal groups were significantly increased （P＞0.05）， and endometrium was also thickened； there was no statistical difference in the changes of body mass， liver and kidney function， liver index， kidney index， the number of leukocyte， lymphocyte and neutrophil in routine blood test among those groups （P＞0.05）， and the morphology of liver and kidney tissue has also no significant difference. However， the number of plasma red blood cells in the progesterone group decreased significantly （P＜0.05）， and there was no statistical significance in the number difference of red blood cells among progesterone-CNA cocrystal groups （P＞0.05）. CONCLUSIONS The progesterone-CNA cocrystal is successfully prepared with good safety in vivo， which significantly improve the solubility of progesterone.

In vivo delivery process and regulating mechanisms of lipid-based nanomedicines / 药学学报

Lipid-based nanocarrier is a classic drug delivery system with great biocompatibility and biodegradability. It can effectively reduce the toxicity of anti-tumor and anti-infective drugs in clinical practice. However, it has not yet met the clinical demand for enhanced therapeutic efficacy, and the clinical application is still very limited. The complex in vivo delivery process of lipid-based nanomedicine and the reciprocal interactions with body lead to unexpected changes in in vivo performance of nanomedicine and seriously hinder clinical translation. Therefore, the in-depth study of the relationships among intrinsic properties of lipid-based nanomedicine, the in vivo delivery process, and the regulatory mechanisms will not only provide guidance for the rational design of nanocarriers, but also promote the clinical translation and precision medicine of new lipid-based nanomedicine. In this review, we summarize the in vivo delivery process, regulating factors and intervention strategies for the in vivo delivery of lipid-based nanomedicine.

Methods of apoptosis detection: advances and applications / 药学学报

Apoptosis, or programmed cell death, is a common phenomenon which involved in a variety of physiological and pathological conditions in humans, such as neurodegenerative diseases, ischemic injury, autoimmune diseases and cancers. Apoptosis can be detected in vitro by morphology, biochemistry, molecular biology, immunology, and other techniques. Probes for cell apoptosis detection in vivo are still under research and various reagents and methods are constantly emerging. However, none of apoptosis detection methods or reagents are perfect and they all have advantages and disadvantages, as well as suitable scope of application. With the increasing application of apoptosis detection techniques, researchers will be confused about how to choose a suitable method to detect apoptosis and define the application range of each apoptosis detection method. Therefore, it is necessary to compare the benefits and drawbacks of existing apoptosis detection techniques as well as their applicable conditions. This article reviews morphological characteristics, molecular mechanism and specific biochemical changes in apoptotic cells. We summarized various apoptosis-detection methods based on these characteristics that can be used in vitro and in vivo, the advantages and disadvantages of each method and the scope of application. Also, we highlighted the existing tracers that have been used in apoptosis detection in vivo, their potentialities and limitations as well as the clinical applications of apoptosis imaging in multiple disease fields.

Comparative study on the methods to determine disintegration time of oral soluble films / 药学学报

Disintegration time is a key parameter that affects the palatability and compliance of oral soluble films. At present, there is no standard method to determine the disintegration time of oral soluble films. In this study, we compared the six methods (pharmacopoeial disintegration method, petri dish method, sponge surface method, slide frame and ball method, partially immersed into liquid (without weight attached) and partially immersed into liquid (with weight attached)) to determine the in vitro disintegration time of oral soluble films with different thickness, and evaluated the correlation with the in vivo disintegration time. The results showed that the repeatability and correlation of pharmacopoeial disintegration method and the partially immersed into liquid method (with weight attached) were excellent, with the endpoint of disintegration testing easy to determine. Partially immersed into liquid method (with weight attached), properly simulating the physiological condition in oral cavity, showed strong operability, good repeatability and in vitro-in vivo correlation, and was suitable for in vitro disintegration evaluation of oral soluble film dosage form. The adult sensory evaluation study was a research-based clinical trial conducted with informed consent from all subjects in accordance with the ethical requirements of Good Clinical Practice.

Preparation of Chinese national potency reference for hepatitis E vaccine / 中国生物制品学杂志

@#ObjectiveTo prepare the hepatitis E vaccine national potency reference for the quality control of hepatitis E vaccine.MethodsA batch of hepatitis E vaccine was selected as the row material,and the screened lyoprotectant was added. After aliquot and freeze-drying,it was prepared as candidate reference material,and the homogeneity and stability were investigated,which was distributed to four laboratories for collaborative calibration and applicability study.Results The 5% trehalose + 2% dextran was selected as the lyoprotectant to prepare the candidate reference material. The aliquot accuracy of candidate reference material was 0. 7%;The coefficient of variation(CV)of antigen content homogeneity was9. 0%;The CV of aluminum content homogeneity was 4. 0%. Moisture content was 1. 7%. The candidate reference material showed good acceleration stability and reconstituted stability. A total of 18 valid data were obtained from the collaborative calibration. The results showed that the average value of median effective dose(ED50)was 0. 15 μg(95% confidence interval of 0. 12 ～ 0. 18 μg)with the intra-laboratory CV of 30. 8% ～ 64. 2%,and the inter-laboratory CV of 32. 6%. Two hepatitis E vaccines produced by two laboratories themselves and the candidate reference material showed good dose-response relationship,of which the seroconversion rate decreased with the decrease of vaccine antigen content and the curve change trend was similar.ConclusionThe first Chinese national potency reference for hepatitis E vaccine(300031-201801)was prepared,which can be used for the quality control of potency ED50detection of hepatitis E vaccine.

Changes of corneal nerve fiber and the correlation analysis with visual acuity in demyelinating optical neuritis patients / 中华眼底病杂志

Objective:To investigate the changes in the nerve fiber layer of the cornea in patients with demyelinating optic neuritis (DON) and its correlation with visual acuity.Methods:A cross-sectional study. From March 2021 to July 2022, 27 cases (39 eyes) of DON patients diagnosed in the Department of Neurology and Ophthalmology of Beijing Tongren Hospital Affiliated to Capital Medical University were enrolled in this study. According to the serological test results, the patients were divided into aquaporin 4 antibody associated optic neuritis (AQP4-ON group) and myelin oligodendrocyte glycoprotein antibody associated optic neuritis (MOG-ON group), with 15 cases (19 eyes) and 12 cases (20 eyes) respectively. According to previous history of glucocorticoid treatment, the patients were divided into glucocorticoid treated group and non-glucocorticoid treated group, with 17 cases (27 eyes) and 10 cases (12 eyes) respectively. Twenty healthy volunteers (20 eyes) with age- and gender-matched were selected as the control group. All eyes underwent best corrected visual acuity (BCVA) and in vivo confocal microscopy (IVCM) examinations. BCVA was performed using Snellen's standard logarithmic visual acuity chart, which was converted into logarithmic minimum angle resolution (logMAR) visual acuity during statistics. The corneal nerve fiber length (CNFL), corneal nerve fiber density (CNFD), corneal nerve fiber branch length (CNBL), corneal nerve fiber branch density (CNBD) and the density of corneal dendritic cells (DC) were detected by IVCM examination. Parameter comparison between groups by t-test and Kruskal-Wallis rank sum test. The correlation between logMAR BCVA and pamameters of corneal nerve fibers were analyzed using Spearman analysis. Results:The CNFL, CNFD, and CNBL of the DON group and the control group were (10.67±2.55) mm/mm 2, (57.78±12.35) root/mm 2, (3.27±1.34) mm/mm 2, and (13.74±3.05) mm/mm 2, (70.95±13.14) root/mm 2, and (4.22±1.03) mm/mm 2, respectively; the difference in CNFL, CNFD, and CNBL between the two groups were statistically significant ( t=4.089, 3.795, 2.773; P<0.05). The CNFL, CNBL, and CNBD of the affected eyes in the MOG-ON group and AQP4-ON group were (12.02±2.13) mm/mm 2, (3.80±1.19) mm/mm 2, (47.97±8.86) fibers/mm 2, and (9.25±2.19) mm/mm 2, (2.72±1.19) mm/mm 2, (39.43±13.86) fibers/mm 2, respectively; the differences in CNFL, CNBL, and CNBD between the two groups were statistically significant ( t=-4.002, -2.706, -2.306; P<0.05). The corneal DC density of the patients in the hormone treated group and the non-hormone treated group was (24.43±8.32) and (41.22±9.86) cells/mm 2, respectively. The difference in corneal DC density between the two subgroups was statistically significant ( P<0.001). Correlation analysis showed that there was a significant negative correlation between logMAR BCVA and CNBL and CNFL in patients with DON ( r=-0.422, -0.456; P<0.05). Conclusions:There are different degrees of corneal nerve fiber damage in patients with different types of DON. There was a negative correlation between BCVA and the length of corneal nerve fibers.

Establishment of an in vivo neutralization model based on H5N1 pseudovirus / 中华微生物学和免疫学杂志

Objective:To establish an in vivo infection model of H5N1 pseudovirus and to detect the neutralizing activity of FHA3 antibody using this model. Methods:Based on the sequence information of hemagglutinin (HA) and neuraminidase (NA) of A/Anhui/1/2005/H5N1 strain, two recombinant plasmids of pcDNA3.1-HA5 and pcDNA3.1-NA1 were constructed. The two plasmids and plasmid pNL4-3.Luc.R-E- were co-transfected into 293T cells to prepare H5N1 pseudovirus supernatant. The morphology of pseudovirus particles in the supernatant was observed by electron microscopy. MDCK cells were infected with the pseudovirus supernatant and the virus titer was detected. BALB/c mice were injected with the pseudovirus supernatant by intraperitoneal injection and subjected to bioluminescence imaging at 2, 5, 8, and 12 d after infection to detect the pseudovirus infection in vivo. The functional activity of FHA3 antibody in vivo was evaluated using the established mouse infection model. Results:The recombinant plasmids pcDNA3.1-HA5 and pcDNA3.1-NA1 were correctly constructed and could be used to prepare pseudovirus supernatants of high titer by co-transfecting 293T cells with the plasmid pNL4-3.Luc.R-E-. The virus particles were round under electron microscope. H5N1 pseudovirus-infected mice exhibits strong fluorescence signals, which were attenuated by FHA3 treatment before challenge.Conclusions:The in vivo infection model of H5N1 pseudovirus was successfully constructed and FHA3 antibody was proved to be protective against the pseudovirus infection.

Clinical application of radiotherapy based on UIH linear accelerators to in vivo dose verification / 中华放射医学与防护杂志

Objective:To explore the clinical application of the electronic portal imaging device (EPID) based on the linear accelerator produced by Shanghai United Imaging Healthcare Co., Ltd. (UIH) to in vivo dose verification. Methods:A total of 68 patients (32 cases with head and neck tumors, 16 cases with chest tumors, and 20 cases with abdomen and pelvis tumors) who were treated with volumetric modulated arc therapy (VMAT) in the Henan Provincial People′s Hospital were selected in this study. Each patient underwent the pre-treatment dose verification using an Arccheck device (Pre Arccheck), the pre-treatment dose verification using an EPID (Pre EPID), and the in vivo dose verification using an EPID (In vivo EPID). Moreover, the position verification based on fan beam computed tomography (FBCT) was also performed for each patient in the first three treatments and then once a week. The patients were treated when the setup error in any direction ( x: left-right, y: head-foot, z: vertical) was less than 3 mm; otherwise, position correction would be conducted. The three-dimensional setup deviation d was calculated according to setup errors x, y, and z. Results:The γ passing rates of dose verifications Pre EPID and In vivo EPID of 68 patients were (99.97±0.1)% and (94.15±3.84)%, respectively, significantly different from that (98.86±1.48)% of the Pre Arccheck dose verification ( t = -6.12, 9.43; P < 0.05). The γ passing rates of the chest, abdomen and pelvis, and head and neck in the In vivo EPID dose verification showed no significant differences ( P > 0.05). The difference in the γ passing rates (5.56±3.72)% between dose verifications Pre EPID and first In vivo EPID was unrelated to the three-dimensional setup deviation d (1.46±1.51 mm) ( P > 0.05). As the treatment proceeded, the γ passing rate of In vivo EPID gradually decreased from (94.15±3.84)% in the first week to (92.15±3.24)% in the fifth week. From the third week to the fifth week, the γ passing rates of In vivo EPID were significantly different from those in the first week ( t = 2.48, 2.75, 3.09, P < 0.05). Conclusions:The setup errors within 3 mm do not affect the γ passing rate of in vivo dose verification. The clinically acceptable threshold for the γ passing rate of in vivo EPID needs to be further determined. In addition, in vivo dose verification can support the clinical application of adaptive radiotherapy to a certain extent.

Solid-phase microextraction of endogenous metabolites from intact tissue validated using a Biocrates standard reference method kit / 药物分析学报

Improved analytical methods for the metabolomic profiling of tissue samples are constantly needed.Currently,conventional sample preparation methods often involve tissue biopsy and/or homogenization,which disrupts the endogenous metabolome.In this study,solid-phase microextraction(SPME)fibers were used to monitor changes in endogenous compounds in homogenized and intact ovine lung tissue.Following SPME,a Biocrates AbsoluteIDQ assay was applied to make a downstream targeted metab-olomics analysis and confirm the advantages of in vivo SPME metabolomics.The AbsoluteIDQ kit enabled the targeted analysis of over 100 metabolites via solid-liquid extraction and SPME.Statistical analysis revealed significant differences between conventional liquid extractions from homogenized tissue and SPME results for both homogenized and intact tissue samples.In addition,principal component analysis revealed separated clustering among all the three sample groups,indicating changes in the metabolome due to tissue homogenization and the chosen sample preparation method.Furthermore,clear differences in free metabolites were observed when extractions were performed on the intact and homogenized tissue using identical SPME procedures.Specifically,a direct comparison showed that 47 statistically distinct metabolites were detected between the homogenized and intact lung tissue samples(P＜0.05)using mixed-mode SPME fibers.These changes were probably due to the disruptive homogenization of the tissue.This study's findings highlight both the importance of sample preparation in tissue-based metabolomics studies and SPME's unique ability to perform minimally invasive extractions without tissue biopsy or homogenization while providing broad metabolite coverage.

In vivo confocal microscopic observations of 49 patients with posterior polymorphous corneal dystrophy / 国际眼科杂志(Guoji Yanke Zazhi)

AIM: To explore the imaging features of 49 patients with posterior polymorphous corneal dystrophy(PPCD)by in vivo confocal microscopy(IVCM).METHODS: Retrospective case series study. A total of 49 patients(86 eyes), including 32 males and 17 females diagnosed with PPCD between January 2013 and January 2021 were collected. The mean age was 42.5±22.9 years. All patients were scanned by IVCM to analyze the density of corneal endothelial cells and described IVCM characteristics of different types of PPCD.RESULTS: The number of endothelial cells in the lesion area of all patients was lower than that in the peripheral area. Under IVCM, 44 eyes(51%)were categorized into type 1 PPCD(vesicular lesions), characterized by single or multiple, central round or irregular crater-like lesion on paracentral corneal endothelial layer; 16 eyes(19%)were categorized into type 2 PPCD(band lesions), which displayed curved and raised edge with scattered or banded-distributed gutta-like lesion between edges. Type 3 PPCD(diffuse lesion)were in 26 eyes(30%), which showed that endothelial cells were missing in many areas. The blurred images of endothelium in most areas featured with spikes lined in a streak, and the clear images in some areas featured with a band lesions. Two patients were followed up for 4-5a. The IVCM images showed different lesions, including the decrease of central corneal endothelial cell density and the iron deposit in the corneal epithelium, etc.CONCLUSION: IVCM is able to scan the characteristic microstructural alterations at the level of endothelium and Descemet membrane in patients with PPCD, and provide an effective image diagnosis for PPCD.