Acute toxicity, 28-day repeated-dose toxicity and toxicokinetic study of timosaponin BII in beagle dogs.

The safety evaluation of timosaponin BII (TBII) in beagle dogs with toxicokinetic study was performed. For the acute oral toxicity study, the minimum lethal dose (MLD) of TBII was more than 2000 mg/kg and suggested the characteristics of absorption saturation. For the 28-day repeated dose oral toxicity and toxicokinetic studies, there was no significant effect on all test parameters except for prolonged APTT in the 60 and 180 mg/kg groups, which recovered after withdrawal. The increase of drug exposure of 180 mg/kg group was not proportional to the increase of administration dose, showing the characteristics of absorption saturation.

Application of preoperative computed tomographic lymphography for precise sentinel lymph node biopsy in breast cancer patients.

BACKGROUND: In light of the extensive application of sentinel lymph node biopsy (SLNB) in clinically node-negative breast cancer patients and the recently investigated failure of SLNB after lumpectomy, it has become important to explore methods for preoperative mapping of sentinel lymph nodes (SLNs) and their lymphatics to direct precise SLNB and improve the identification rate of SLNs. METHODS: Twenty-seven patients with suspected breast cancer based on the results of the clinical examination and imaging were enrolled in the study. Computed tomographic lymphography (CTLG) followed by CT three-dimensional reconstruction was performed to determine the localization of SLNs and lymphatics on the body surface preoperatively. Intraoperatively combined staining with methylene blue and indocyanine green was used to evaluate the accuracy and feasibility of CTLG. RESULTS: SLNs and lymphatics from the breast were identified using CTLG in all patients, and preoperative SLNs and lymphatics localization on the body surface showed a significant role in the selection of operative incision and injection points. The accuracy rate of SLN and lymphatic detection by CTLG was 92.6% compared with intraoperatively combined staining. Moreover, preoperative CTLG performed well in SLN number detection, and the accuracy rate was 95.2%. CONCLUSION: We evaluate the procedure and application of preoperative CTLG in the superficial localization of SLNs and lymphatics, which may lead to a decreased incidence of cutting off the lymphatics of SLNs and consequently more rapid and accurate SLN detection. This method promotes personalized SLN mapping, providing detailed information about the number and anatomical location of SLNs and lymphatics for adequate surgical planning for breast cancer patients.

Identification and characterization of terpene synthase genes accounting for volatile terpene emissions in flowers of Freesia x hybrida.

The development of flower scents was a crucial event in biological evolution, providing olfactory signals by which plants can attract pollinators. In this study, bioinformatics, metabolomics, and biochemical and molecular methodologies were integrated to investigate the candidate genes involved in the biosynthesis of volatile components in two cultivars of Freesia x hybrida, Red River® and Ambiance, which release different categories of compounds. We found that terpene synthase (TPS) genes were the pivotal genes determining spatiotemporal release of volatile compounds in both cultivars. Eight FhTPS genes were isolated and six were found to be functional: FhTPS1 was a single-product enzyme catalyzing the formation of linalool, whereas the other four FhTPS proteins were multi-product enzymes, among which FhTPS4, FhTPS6, and FhTPS7 could recognize geranyl diphosphate and farnesyl diphosphate simultaneously. The FhTPS enzymatic products closely matched the volatile terpenes emitted from flowers, and significant correlations were found between release of volatile terpenes and FhTPS gene expression. Graphical models based on these results are proposed that summarize the biosynthesis of Freesia floral volatile terpenes. The characterization of FhTPS genes paves the way to decipher their roles in the speciation and fitness of Freesia, and this knowledge could also be used to introduce or enhance scent in other plants.

Acute toxicity, 28-day repeated-dose toxicity and toxicokinetic study of timosaponin BII in rats.

Timosaponin BII (TBII), a major steroidal saponin isolated from Anemarrhena asphodeloides Bge., displays a variety of promising pharmacological activities, such as neuroprotection, enhancement of learning and memory, vascular protection and inhibition of platelet aggregation; therefore, it has been developed as a pharmaceutical for prevention or treatment of dementia. Given the safety concerns surrounding timosaponins and the absence of studies on the safety of TBII, the potential toxicity of TBII was evaluated in toxicity and toxicokinetic studies in rats. In the acute oral toxicity study, loose stools were observed in rats receiving 4000 mg/kg, and the symptoms recovered within 1 day. In the 28-day repeated-dose oral toxicity and toxicokinetic study, rats receiving 540 mg/kg showed loose stools and a slight deceleration of body weight growth in both sexes, and the females also showed a slight decrease in food consumption. Moreover, urinalysis indicated reversible treatment-related toxicity in rats receiving 540 mg/kg. The toxicokinetic study demonstrated a dose-dependent increase in systematic exposure to TBII after 28 successive days of oral treatment with TBII. The accumulation coefficients of TBII were 4.35, 1.70 and 1.81, respectively, in rats that received 60, 180 and 540 mg/kg. The no-observed-adverse-effect level (NOAEL) is proposed to be 180 mg/kg.

Ovatodiolide inhibited hepatocellular carcinoma stemness through SP1/MTDH/STAT3 signaling pathway.

Hepatocellular carcinoma (HCC) is characterized with high recurrence and mortality, and the clinical treatments for HCC are very limited. Hepatocellular carcinoma stem cells are the root of HCC progress, recurrence, and multidrug resistance. Ovatodiolide (OVA) is a bioactive diterpenoid served as an inflammatory and immunotherapeutic responses modulator. In this research, we found OVA inhibited HCC stemness through inhibiting MTDH gene transcription. Moreover, we firstly discovered transcription factor SP1 bound to the promoter region of MTDH to transcriptionally regulate MTDH level. Mechanically, we demonstrated OVA decreased SP1 protein stability to transcriptionally inhibit MTDH gene, and inhibited the nuclear translocation of p65, and then diminished IL-6 level to suppress JAK/STAT3 signaling pathway, eventually decreases CD133 level and the stemness of HCC. Furthermore, we demonstrated ACT004, OVA derivative with high metabolic stability towards cytochrome P450 enzymes, showed no genotoxicity and no accumulative or delayed toxicities after long-term administration in rats. And the in vivo efficacy experiments indicated ACT004 inhibited tumor growth of hepatocellular carcinoma. In conclusion, we revealed the mechanism of OVA in regulating HCC stemness, detected the toxicity of OVA derivative and evaluated the in vivo efficacy which lays a foundation for further discovery of anti-HCC stem cell agents and provide a new strategy for the application of OVA in clinical treatment.

[Determination of 2, 4-toluenediamine in urine by gas chromatography].

OBJECTIVE: To establish a method for determining the content of 2,4-toluenediamine, a urinary metabolite of toluene diisocyanate, by gas chromatography. METHODS: Urine samples were collected, and acidification, extraction, derivatization, separation with a capillary column, and detection with an electron capture detector were performed. The target compound was qualified by retention time and quantified by peak area. RESULTS: The concentration of 2, 4-toluenediamine showed a linear relationship with peak area within 0.0∼40 ng/ml, with a correlation coefficient 0.9995; the limit of detection was 0.44 ng/ml, the lower limit of quantification was 1.47 ng/ml, the relative standard deviation was 1.85%∼4.05%; the recovery rate was 97.98%∼99.28%. CONCLUSION: The method has the advantages of high sensitivity and high accuracy and can be used for determination of 2, 4-toluenediamine in urine.

[Effect of toluene diisocyanate on lung function of workers].

OBJECTIVE: To investigate the effect of long-term exposure to toluene diisocyanate (TDI) on the lung function of TDI-exposed workers. METHODS: A factory was selected for this occupational epidemiological investigation. The workers who were exposed to TDI and had complete physical examination records in recent 3 years were the exposed group (n = 45), while the company's administrative staff, logistics staff, and other non-TDI-exposed workers who had complete physical examination records in recent 3 years were the control group (n = 47). The two groups were compared in terms of lung function indices. RESULTS: Compared with the control group, the 2009 exposure group had significantly lower forced expiratory volume in one second (FEV1.0), FEV1.0/forced vital capacity (FVC), and maximal expiratory flow at 25% of FVC (MEF25) (P < 0.05), the 2010 exposure group had significantly lower FEV1.0, FEV1.0/FVC,maximum voluntary ventilation (MVV), and maximal expiratory flow at 50% of FVC (MEF50) (P < 0.05), and the 2011 exposure group had significantly lower FEV1.0, FEV1.0/FVC, peak expiratory flow (PEF), MEF25, and MEF50 (P < 0.05). CONCLUSION: Long-term exposure to TDI can lead to certain impairment of lung function in workers, which may be reflected by decreased lung function indices such as vital capacity, FVC, FEV1.0, FEV1.0/FVC, PEF, and MVV.

Preclinical safety evaluation of a bivalent inactivated EV71-CA16 vaccine in mice immunized intradermally.

Hand, foot and mouth disease is a common acute viral infectious disease that poses a serious threat to the life and health of young children. With the development of an effective inactivated EV71 vaccine, CA16 has become the main pathogen causing HFMD. Effective and safe vaccines against this disease are urgently needed. In our previous study, a bivalent inactivated vaccine was shown to have good immunogenicity and to induce neutralizing antibodies in mice and monkeys. Repeated administration toxicity is a critical safety test in the preclinical evaluation of vaccines. In this study, BALB/c mice were used to evaluate the toxicity of the bivalent vaccine after multiple intradermal administrations. Clinical observation was performed daily, and body weight, food intake, hematological characteristics, serum biochemical parameters, antinuclear antibodies, CD4+/CD8a+ T-cell proportions, bone marrow smear results and pathology results were recorded. The results showed that there was no significant change at the injection site and no adverse reactions related to the vaccine. The bivalent inactivated EV71-CA16 vaccine exhibits good safety in mice, and these results provide a sufficient basis for further clinical trials.

Safety evaluation of revefenacin at the approved dose in patients with chronic obstructive pulmonary disease: A meta-analysis.

BACKGROUND: Revefenacin is the first once-daily long-acting muscarinic antagonist (LAMA) for nebulization use in maintenance therapy for patients with chronic obstructive pulmonary disease (COPD). OBJECTIVE: To investigate the safety and tolerability profile of revefenacin at the approved dose (175 µg), compared with placebo and a lower dose (88 µg), for the treatment of COPD. METHODS: Available randomized controlled trials (RCTs), both published and unpublished, were identified via databases. Risk differences (RDs) and risk ratios (RRs), with their corresponding 95% confidence intervals (CIs) were calculated as effect sizes. RESULTS: One unpublished RCT and four articles containing 5 RCTs were included. Combined results showed that there were no significant differences between COPD patients receiving 175 µg revefenacin and those receiving a placebo, concerning the risk of discontinuation due to adverse events (AEs), any all-grade AE, or any serious AE. 175 µg revefenacin also did not significantly increase the risk of antimuscarinic-related AEs, cardiovascular AEs, or 12 commonly reported AEs. Plus, a lower dose of 88 µg was shown to share a comparable safety profile with the 175 µg revefenacin. A non-significant trend towards a decrease in risks of AEs for 175 µg revefenacin was observed. The most frequently reported AE for each group was COPD worsening/exacerbation. CONCLUSION: Revefenacin at the approved dose is generally well-tolerated and safe with minimal AEs, which supports its use as a once-daily nebulized LAMA for the treatment of moderate to severe stable COPD. Additional studies are needed to complete the safety and tolerability profile.

Preclinical safety assessment of a combined vaccine against Hepatitis a virus and enterovirus 71.

Since 2007, Hepatitis A (HAV) vaccination has been a part of the National Immunization Program of China. Recognizing enterovirus 71 (EV71) as the most important pathogen in severe hand, foot and mouth disease, an inactivated EV71 vaccine was successfully marketed in 2015. Based on the concept of one vaccine preventing two diseases and owing to similarities in vaccine preparation and the overlap of the eligible population, a combination of the inactivated HAV vaccine and inactivated EV71 vaccine is theoretically feasible and desirable. However, the optimal vaccinationschedule for this combination vaccine has yet to be optimized. Use of this combined vaccine would not only decrease the number of vaccinations, but also lower associated cost. This study aimed to investigate the toxicity and adverse reactions of the combined HAV-EV71 vaccine under Good Laboratory Practice conditions to provide a reference for clinical studies/applications in the future. CD®(Sprague Dawley) IGS rats were employed for single-dose toxicity testing using a high dose, and repeated-dose toxicity testing using high, as well as low doses. Animals that received only a single dose showed no obvious clinical symptoms nor abnormal body weight, and no significant gross pathological change at the experimental endpoint at necropsy. In the rats injected with three doses, phagocytosis of basophilic granules by macrophages was observed in the inguinal, mesenteric, and local lymph nodes, besides irritation at the administration site. At 56 days after the last dose, no significant histopathological change was observed in the lymph nodes, and local irritation gradually faded. Further, systematic allergy testing was performed in guinea pigs. After systemic sensitization and challenge with the HAV-EV71 vaccine, animals showed normal weight gain and no allergic reactions. This study, therefore, confirmed a good safety profile of the inactivated HAV and EV71 combined vaccine.

Preclinical evaluation of recombinant HFMD vaccine based on enterovirus 71 (EV71) virus-like particles (VLP): Immunogenicity, efficacy and toxicology.

Enterovirus 71 (EV71) is one of the major causative agents for hand, foot and mouth disease (HFMD) in children. Currently, three inactivated EV71 vaccines have been approved by Chinese government. We previously demonstrated that recombinant EV71 virus-like particles (VLP) produced in Pichia pastoris can be produced at a high yield with a simple manufacturing process, and the candidate vaccine elicited protective humoral immune responses in mice. In present study, the nonclinical immunogenicity, efficacy and toxicity of the EV71 vaccine was comprehensively evaluated in rodents and non-human primates. The immunogenicity assessment showed that EV71 VLPs vaccine elicited high and persistent neutralizing antibody responses, which could be comparable with a licensed inactivated vaccine in animals. The immune sera of vaccinated mice also exhibited cross-neutralization activities to the heterologous subtypes of EV71. Both passive and maternal antigen specific antibodies protected the neonatal mice against the lethal EV71 challenge. Furthermore, nonclinical safety assessment of EV71 VLP vaccine showed no signs of systemic toxicity in animals. Therefore, the excellent immunogenicity, efficacy and toxicology data supported further evaluation of the VLP-based EV71 vaccine in humans.

Cloning and functional characterization of a carotenoid cleavage dioxygenase 2 gene in safranal and crocin biosynthesis from Freesia hybrida.

Safranal and crocin, commonly derived from the oxidative cleavage reaction of zeaxanthin in plants, are two kinds of apocarotenoids with versatile functions, which were only found in limited number of plant species. In this study, both metabolites were detected and varied concomitantly with the expression of carotenoid cleavage dioxygenase (CCD) genes in Freesia hybrida, Red River® and Ambiance cultivars. The newly isolated CCD, denoted here as FhCCD2, was phylogenetically clustered with other reported saffron CCD2s. Besides, ten introns were also observed in the genomic DNA sequence of FhCCD2 and the presence of N-terminal transporter peptide suggested its plastidial sub-localization. Biochemical analysis showed that the FhCCD2 cleaved zeaxanthin at the 7, 8 and 7', 8' double bonds to generate intermediates prerequisite for the biosynthesis of safranal and crocin. Further, gene transient expression analysis showed that the promoter of FhCCD2 was functional in Ambiance as well as Red River® cultivars, even with slight variation in their promoter sequence. At present, CCD2 proteins have only been found in Freesia and Crocus genus of Iridaceae family. Phylogenetic and intron position analysis infer that CCD2 perhaps emerged after the intron loss during evolutionary process of CCD1 or their shared ancestry.

Relationship between Upper Extremity Lymphatic Drainage and Sentinel Lymph Nodes in Patients with Breast Cancer.

PURPOSE: The purpose of this study was to identify the relationship between upper extremity lymphatics and sentinel lymph nodes (SLNs) in breast cancer patients. METHODS: Forty-four patients who underwent axillary reverse mapping (ARM) during axillary lymph node dissection (ALND) with SNL biopsy (SLNB) between February 2017 and October 2017 were investigated. ARM was performed using indocyanine green (ICG) to locate the upper extremity lymphatics; methylene blue dye was injected intradermally for SLN mapping. RESULTS: ARM nodes were found in the ALND fields of all examined patients. The rate of identification of upper extremity lymphatics within the SLNB field was 65.9% (29 of 44). The ARM nodes were involved in metastases arising from primary breast tumors in 7 of the patients (15.9%), while no metastases were detected in pathologic axillary lymph node-negative patients. Lymphatics from the upper extremity drained into the SLNs in 5 of the 44 patients (11.4%); their ARM-detected nodes were found to be in close proximity to the SLNs. CONCLUSIONS: The ARM nodes and SLNs are closely related and share lymphatic drainage routes. The ARM procedure using fluorescence imaging is both feasible and, in patients who are SLN negative, oncologically safe. ARM using ICG is therefore effective for identifying and preserving upper extremity lymphatics, and SLNB combined with ARM appears to be a promising surgical refinement for preventing upper extremity lymphoedema. CLINICAL TRIAL REGISTRATION: This trial is registered with ClinicalTrial.gov: NCT02651142.

DNA damaging effects of carbofuran and its main metabolites on mice by micronucleus test and single cell gel electrophoresis.

The DNA damaging effects of the carbamate pesticide carbofuran and its four metabolites (carbofuranphenol, 3-ketocarbofuran, 3-hydrocarbofuran and nitrosocarbofuran) on mice were evaluated by single cell gel electrophoresis (SCGE) assay and micronucleus test. KM mice were exposed to test compounds with different doses of 0.1, 0.2 and 0.4 mg/kg through intraperitoneal injection two times with an internal of 24 h, and then killed by cervical dislocation 6 h after the second injection. In SCGE assay, isolated mice peripheral blood lymphocytes were employed to determine DNA damaging degree after a 1 h treatment by test compounds and a following electrophoresis. Carbofuran and carbofuranphenol showed negative results in both test and had no obvious toxicity. 3-Hydrocarbofuran and nitrosocarbofuran were positive. 3-Ketocarbofuran could not induce micronucleus formation but caused significant DNA migration in SCGE test. These tests revealed that 3-ketocarbofuran, 3-hydrocarbofuran and nitrosocarbofuran are potential mutagesis and further research is needed.

Identification of antibacterial and antioxidant constituents of the essential oils of Cynanchum chinense and Ligustrum compactum.

The aim of this research was to determine the chemical composition, antioxidant and antibacterial properties of the essential oils from Cynanchum chinense and Ligustrum compactum and isolation of antioxidant and antibacterial constituents from the essential oils. Thirty-eight components were identified in essential oils. Based on bioactivity-guided fractionation, guaiacol, linalool and 2-phenylethanol were isolated and identified as active constituents. Both L. compactum flower oil and 2-phenylethanol showed high antibacterial performance, with inhibition zone from 22.8 ± 0.8 to 11.9 ± 2.0 mm at highest concentration, and minimum inhibitory concentration values ranging from 0.25% to 1%. In both DPPH and ABTS assay, the active constituent guaiacol (IC50 = 4.15 ± 0.72 and 9.12 ± 0.98 µg mL(-1), respectively) exhibited high antioxidant activity, and the oils showed moderate antioxidant activity. These results indicate potential efficacy of active constituents and essential oils of L. compactum and C. chinense to control food-borne pathogenic and spoilage bacteria.

Dynamics of fipronil residue in vegetable-field ecosystem.

Fipronil insecticide has been widely used to control vegetable pests in China recently. The research was conducted to evaluate the fate of fipronil in vegetable-field ecosystem and provide the scientific basis of using this insecticide. Developed on the analytical methods of fipronil residue and its four metabolisms, the degradation dynamics of their residue in a vegetable and the soil of the vegetable fields was studied. The results showed that (1) degradation of fipronil was faster in pakchoi (half-life 2.6 days) than in soil (half-life 7.3 days); (2) degradation reaction occurred in soil was governed mainly by photodegradation and oxidization accompanying with production of the metabolites, MB46513 and MB46136. Reduction and hydrolyzation played little role in the degradation process. In pakchoi, degradation was mainly contributed by reduction though oxidization and hydrolyzation occurred simultaneously. The metabolite products were MB45950, MB46136 and RPA200766; (3) the final residue in pakchoi was at a level of 0.003 mg kg(-1), which was much lower than the USA's upper limit of 0.04 mg kg(-1) in rice. Therefore, a dosage of 24 g hm(-2) was suggested and considered as safe to human beings and animals.

[Immunity of peritoneal monocytes against Plasmodium yoelii infected erythrocytes].

OBJECTIVE: To test the immunity of peritoneal monocytes against Plasmodium yoelii infected red blood cells (target cells). METHODS: Saponinized Plasmodium yoelii infected red blood cells (SPRBC, Ghost erythrocyte) were used to immunize mice i.p twice. Three weeks later, the infected red blood cells were injected i.p.; 90 min later, the total peritoneal cells were isolated and washed for scanning electromicroscopy to observe the effects of the peritoneal monocyte to the target cell. RESULTS: The peritoneal cells of the immunized mice were activated after 90 min of the challenge of target cells. The size of the cell was not even and the pili on the cell surface turned to be long and densed. Cell interconnections were found among the cells. In some peritoneal monocytes, their cell plasma were scattered (omlette-like) or with the shape as "cellular bomb". The scattered or the sheeted pili and spredding cell plasma could adhere to the target cells which were perforated densely and damaged. CONCLUSION: The protective adaptive immunity exists in the peritoneal monocytes of immunized mice.