骨质疏松疫苗载体:Qβ病毒样颗粒的表达、鉴定和纯化

 目的 优化表达和纯化方式,制备出具有正确空间结构且纯度和产量较高的骨质疏松疫苗载体Qβ病毒样颗粒(Qβvirus-like particles,Qβ-VLPs)。方法 在基因合成时删除A1基因序列中Qβ衣壳蛋白终止密码子之后的序列,只合成Qβ衣壳蛋白的基因序列。将Qβ衣壳蛋白基因序列克隆到pET30a载体质粒上,用BL21(DE3)、BL21(DE3)pLysS、Rosetta(DE3)三种不同菌株进行蛋白表达,然后用SDS-PAGE和透射电镜验证是否表达出Qβ-VLPs的正确结构。设置2个蛋白表达诱导剂IPTG( Isopropyl β-D-1-thiogalactopyranoside,异丙基-β-D-硫代半乳糖苷)浓度和3种菌体破碎方式,分别优化蛋白表达条件和菌体破碎方案,然后将得到的Qβ-VLPs上清液通过硫酸铵聚沉、高速离心和分子筛分选进行Qβ-VLPs的纯化。结果 仅BL21(DE3)pLysS菌株可以表达出SDS-PAGE中呈现5-6聚体、透射电镜下呈现25~30 nm球形结构的Qβ-VLPs。使用0.5 mM浓度的IPTG表达的Qβ-VLPs产量较0.2 mM时高2.8倍,使用超声破碎菌体的方式可以获得较高的蛋白分离效率。将Qβ-VLPs上清液通过本实验过程中的方案进行纯化后纯度可达90%左右。结论 该实验探索出具有正确空间结构、组成单一且纯度较高的Qβ-VLPs的制备方案,为相关疫苗制备和研究奠定基础。     

Factors associated with Q fever vaccination in Australian wildlife rehabilitators

Australian wildlife rehabilitators (AWR) are at risk of contracting Q fever, a serious zoonotic disease caused by Coxiella burnetii. Despite Australian government recommendations for AWR to receive Q fever vaccination (QFV), and the availability of a safe and effective vaccine in Australia, shortfalls in vaccine uptake have been observed in AWR. This study aimed to determine factors associated with QFV status and describe AWR attitudes and potential barriers towards QFV. Data were obtained from a nationwide, online, cross-sectional survey of AWR undertaken in 2018. Approximately-three quarters (200/265; 75.5 %) of those that had heard of Q fever were also aware of the Q fever vaccine, and of those, 25.5 % (51/200) were vaccinated. Barriers to QFV, among unvaccinated respondents who had also heard of Q fever and the vaccine (149/200; 74.5 %), included concerns regarding the safety, efficacy, and importance of the Q fever vaccine. Complacency toward vaccination, convenience of vaccination, and a lack of Q fever knowledge were also notable barriers. Only 27.7 % (41/148) of respondents reported having had vaccination recommended to them. Multivariable logistic regression identified that vaccinated AWR were more likely to be aged ≤ 50 years (OR 4.51, 95 % CI: 2.14–10.11), have had a university level education (OR 2.78, 95 % CI: 1.39–5.73), have resided in New South Wales/Australian Capital Territory and Queensland than in other Australian jurisdictions (OR 2.9, 95 % CI: 1.10–8.83 and OR 4.82, 95 % CI: 1.64–16.00 respectively) and have attended an animal birth (OR 2.14, 95 % CI: 1.02–4.73). Knowledge gaps regarding Q fever and QFV in AWR demonstrated the need for interventions to raise the awareness of the potential health consequences of C. burnetii exposure and Q fever prevention. Education programs to allow AWR to develop an informed perspective of Q fever and QFV, coupled with improvements in vaccine affordability and the implementation of programs to enhance accessibility, may also increase vaccine uptake.     

N-Acetylaspartate in the CNS: from neurodiagnostics to neurobiology

The brain is unique among organs in many respects, including its mechanisms of lipid synthesis and energy production. The nervous system-specific metabolite N-acetylaspartate (NAA), which is synthesized from aspartate and acetyl-coenzyme A in neurons, appears to be a key link in these distinct biochemical features of CNS metabolism. During early postnatal central nervous system (CNS) development, the expression of lipogenic enzymes in oligodendrocytes, including the NAA-degrading enzyme aspartoacylase (ASPA), is increased along with increased NAA production in neurons. NAA is transported from neurons to the cytoplasm of oligodendrocytes, where ASPA cleaves the acetate moiety for use in fatty acid and steroid synthesis. The fatty acids and steroids produced then go on to be used as building blocks for myelin lipid synthesis. Mutations in the gene for ASPA result in the fatal leukodystrophy Canavan disease, for which there is currently no effective treatment. Once postnatal myelination is completed, NAA may continue to be involved in myelin lipid turnover in adults, but it also appears to adopt other roles, including a bioenergetic role in neuronal mitochondria. NAA and ATP metabolism appear to be linked indirectly, whereby acetylation of aspartate may facilitate its removal from neuronal mitochondria, thus favoring conversion of glutamate to alpha ketoglutarate which can enter the tricarboxylic acid cycle for energy production. In its role as a mechanism for enhancing mitochondrial energy production from glutamate, NAA is in a key position to act as a magnetic resonance spectroscopy marker for neuronal health, viability and number. Evidence suggests that NAA is a direct precursor for the enzymatic synthesis of the neuron specific dipeptide N-acetylaspartylglutamate, the most concentrated neuropeptide in the human brain. Other proposed roles for NAA include neuronal osmoregulation and axon-glial signaling. We propose that NAA may also be involved in brain nitrogen balance. Further research will be required to more fully understand the biochemical functions served by NAA in CNS development and activity, and additional functions are likely to be discovered.     

Effect of β-hydroxy-β-methylglutaryl coenzyme a reductase inhibitors and antioxidant vitamins on free radical lipid oxidation in rat liver

We studied the effects of two inhibitors of β-hydroxy-β-methylglutaryl coenzyme A reductase, simvastatin and lovastatin, on the lag phase of ascorbate-dependent lipid oxidation in rat liver. Oxidizability of liver biological membranes significantly increased in intact animals and rats with induced hypercholesterolemia after peroral administration of these statins. The lag phase of ascorbate-dependent lipid oxidation in liver biomembranes decreased by 2.1 times in hypercholesterolemic rats. In animals of the lovastatin group this parameter decreased by 4.4 times compared to the control. In intact rats receiving simvastatin, the lag phase of oxidation in biomembranes from the liver decreased practically by 2 times. At the same time, in animals receiving simvastatin in combination with antioxidant vitamins (vitamins E and C, provitamin A) and selenium, the period of induction of oxidation increased by 3.3 times. Our results indicate that β-hydroxy-β-methylglutaryl coenzyme A reductase inhibitors produce a prooxidant effect on the liver, which can be prevented by administration of antioxidant agents. __________ Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 143, No. 4, pp. 390–393, April, 2007     

Effect of β-hydroxy-β-methylglutaryl coenzyme a reductase inhibitor atorvastatin on contractility of the isolated rat heart under normal conditions and during oxidative stress

Long-term administration of β-hydroxy-β-methylglutaryl coenzyme A reductase inhibitor atorvastatin to rats was accompanied by an increase in the relative weight of the heart and decrease in the rate of pressure development in the isovolumic heart. During oxidative stress induced by addition of 100 μM H₂O₂ to the perfusate, the decrease in contractile function was more pronounced that in the control. Our results indicate that administration of atorvastatin is accompanied by a decrease in myocardial contractility, which becomes more pronounced under conditions of oxidative stress. __________ Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 143, No. 4, pp. 383–385, April, 2007     

An outbreak of Q fever associated with parturient cat exposure at an animal refuge and veterinary clinic in southeast Queensland

Objective: To determine the source of a Q fever outbreak in humans at an animal refuge and veterinary clinic in southeast Queensland from October to December 2016. Methods: Case interviews and a retrospective cohort study of animal refuge and veterinary clinic staff using a self‐administered questionnaire related to clinical history of Q fever, Q fever vaccination status and workplace activities during the exposure period. Results: Seven cases (six confirmed, one probable) were identified. Forty‐three questionnaires were completed (92% response rate). Workplace activities associated with the greatest risk of illness were the disposal of deceased cats or dogs (RR, 14.0; 95%CI, 1.9–104.1) and participating in euthanasia of cats or dogs (RR, 4.6; 95%CI, 1.3–16.9). Five feline birthing events occurred at the animal refuge from 25 September to 19 October 2016, each with subsequent euthanasia of the queen cat and litter. All cases had likely exposure to a specific queen cat and her litter that were euthanised the same day as the birthing event. Conclusions: A parturient cat was the most likely source of the outbreak. Implications for public health: Occupational groups and others with regular exposure to feline or canine parturient products should receive Q fever vaccine.     

Developing a quality by design approach to model tablet dissolution testing: an industrial case study

Abstract

This study applied the concept of Quality by Design (QbD) to tablet dissolution. Its goal was to propose a quality control strategy to model dissolution testing of solid oral dose products according to International Conference on Harmonization guidelines. The methodology involved the following three steps: (1) a risk analysis to identify the material- and process-related parameters impacting the critical quality attributes of dissolution testing, (2) an experimental design to evaluate the influence of design factors (attributes and parameters selected by risk analysis) on dissolution testing, and (3) an investigation of the relationship between design factors and dissolution profiles. Results show that (a) in the case studied, the two parameters impacting dissolution kinetics are active pharmaceutical ingredient particle size distributions and tablet hardness and (b) these two parameters could be monitored with PAT tools to predict dissolution profiles. Moreover, based on the results obtained, modeling dissolution is possible. The practicality and effectiveness of the QbD approach were demonstrated through this industrial case study. Implementing such an approach systematically in industrial pharmaceutical production would reduce the need for tablet dissolution testing.     

The Effects of Coenzyme Q10 Supplementation on Glucose Metabolism,Lipid Profiles,Inflammation, and Oxidative Stress in Patients With Diabetic Nephropathy: A Randomized,Double-Blind,Placebo-Controlled Trial

Objective: Data on the effects of coenzyme Q10 (CoQ10) supplementation on glucose metabolism, lipid profiles, inflammation, and oxidative stress in subjects with diabetic nephropathy (DN) are scarce. This research was done to determine the effects of CoQ10 supplementation on metabolic status in subjects with DN.

Methods: This randomized double-blind placebo-controlled clinical trial was done in 50 subjects with DN. Participants were randomly assigned into two groups to intake either 100 mg/day CoQ10 supplements (n = 25) or placebo (n = 25) for 12 weeks. Fasting blood samples were obtained at first and after 12-week intervention to quantify metabolic profiles.

Results: After 12 weeks of treatment, compared with the placebo, CoQ10 supplementation resulted in significant decreases in serum insulin levels (?3.4 ± 6.8 vs +0.8 ± 6.4 µIU/mL, p = 0.02), homeostasis model of assessment-estimated insulin resistance (?1.0 ± 2.0 vs +0.2 ± 1.8, p = 0.03), homeostasis model of assessment-estimated B cell function (?12.3 ± 26.3 vs +3.5 ± 23.1, p = 0.02) and HbA1c (?1.1 ± 1.0 vs ?0.1 ± 0.2%, p < 0.001), and a significant improvement in quantitative insulin sensitivity check index (+0.009 ± 0.01 vs ?0.006 ± 0.01, p = 0.01). In addition, CoQ10 supplementation significantly decreased plasma malondialdehyde (MDA) (?0.6 ± 0.5 vs +0.5 ± 1.0 µmol/L, p < 0.001) and advanced glycation end products levels (AGEs) (?316.4 ± 380.9 vs +318.6 ± 732.0 AU, p < 0.001) compared with the placebo. Supplementation with CoQ10had no significant impacts on fasting plasma glucose (FPG), lipid profiles, and matrix metalloproteinase-2 (MMP-2) compared with the placebo.

Conclusions: Taken together, our study demonstrated that CoQ10 supplementation for 12 weeks among DN patients had favorable effects on glucose metabolism, MDA, and AGEs levels, but unchanged FPG, lipid profiles, and MMP-2 concentrations.     

Fatal intoxication by 5F–ADB and diphenidine: Detection,quantification, and investigation of their main metabolic pathways in humans by LC/MS/MS and LC/Q‐TOFMS

Despite the implementation of a new blanket scheduling system in 2013, new psychoactive substance (NPS) abuse remains a serious social concern in Japan. We present a fatal intoxication case involving 5F–ADB (methyl 2‐[1‐(5‐fluoropentyl)‐1H–indazole‐3‐carboxamido]‐3,3‐dimethylbutanoate) and diphenidine. Postmortem blood screening by liquid chromatography/quadrupole time‐of‐flight mass spectrometry (LC/Q‐TOFMS) in the information‐dependent acquisition mode only detected diphenidine. Further urinary screening using an in‐house database containing NPS and metabolites detected not only diphenidine but also possible 5F–ADB metabolites; subsequent targeted screening by LC/tandem mass spectrometry (LC/MS/MS) allowed for the detection of a very low level of unchanged 5F–ADB in postmortem heart blood. Quantification by standard addition resulted in the postmortem blood concentrations being 0.19 ± 0.04 ng/mL for 5F–ADB and 12 ± 2.6 ng/mL for diphenidine. Investigation of the urinary metabolites revealed pathways involving ester hydrolysis (M1) and oxidative defluorination (M2), and further oxidation to the carboxylic acid (M3) for 5F–ADB. Mono‐ and di‐hydroxylated diphenidine metabolites were also found. The present case demonstrates the importance of urinary metabolite screening for drugs with low blood concentration. Synthetic cannabinoids (SCs) fluorinated at the terminal N‐alkyl position are known to show higher cannabinoid receptor affinity relative to their non‐fluorinated analogues; 5F–ADB is no exception with high CB₁ receptor activity and much greater potency than Δ⁹‐THC and other earlier SCs, thus we suspect its acute toxicity to be high compared to other structurally related SC analogues. The low blood concentration of 5F–ADB may be attributed to enzymatic and/or non‐enzymatic degradation, and further investigation into these possibilities is underway.