Preclinical evaluation of a plant-derived SARS-CoV-2 subunit vaccine: Protective efficacy,immunogenicity, safety,and toxicity

Coronavirus disease 2019 (COVID-19) is an acute respiratory illness caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The prevention of SARS-CoV-2 transmission has become a global priority. Previously, we showed that a protein subunit vaccine that was developed based on the fusion of the SARS-CoV-2 receptor-binding domain (RBD) to the Fc portion of human IgG1 (RBD-Fc), produced in Nicotiana benthamiana, and adjuvanted with alum, namely, Baiya SARS-CoV-2 Vax 1, induced potent immunological responses in both mice and cynomolgus monkeys. Hence, this study evaluated the protective efficacy, safety, and toxicity of Baiya SARS-CoV-2 Vax 1 in K18-hACE2 mice, monkeys and Wistar rats. Two doses of vaccine were administered three weeks apart on Days 0 and 21. The administration of the vaccine to K18-hACE2 mice reduced viral loads in the lungs and brains of the vaccinated animals and protected the mice against challenge with SARS-CoV-2. In monkeys, the results of safety pharmacology tests, general clinical observations, and a core battery of studies of three vital systems, namely, the central nervous, cardiovascular, and respiratory systems, did not reveal any safety concerns. The toxicology study of the vaccine in rats showed no vaccine-related pathological changes, and all the animals remained healthy under the conditions of this study. Furthermore, the vaccine did not cause any abnormal toxicity in rats and was clinically tolerated even at the highest tested concentration. In addition, general health status, body temperature, local toxicity at the administration site, hematology, and blood chemistry parameters were also monitored. Overall, this work presents the results of the first systematic study of the safety profile of a plant-derived vaccine, Baiya SARS-CoV-2 Vax 1; this approach can be considered a viable strategy for the development of vaccines against COVID-19.     

靶向测序诊断黑棘皮病1家系

【摘要】 报道临床症状不典型的家族性黑棘皮病1家系。先证者女，4岁，自1周岁时，颈部、腹部出现黑色斑片，近年来逐渐扩大至唇周、躯干前部。腹部皮肤全反式共聚焦显微镜检查可见乳头环下延扭曲及沟壑结构，乳头环内可见中高折光颗粒结构。先证者父亲及祖母既往有类似病史，但随着年龄增长色素沉着自发性消退，仅有局部皮纹增粗。采集先证者及父母、祖母外周血，对先证者外周血DNA行Panel靶向测序，结果显示，先证者存在FGFR3基因14号外显子c.1949A>C（p.Lys650Thr）错义突变，Sanger测序验证证实先证者及其父亲和祖母均存在此突变。诊断：家族性黑棘皮病。     

慢性强迫游泳应激对大鼠情绪和脑细胞外信号调节激酶磷酸化水平的影响

目的探讨慢性强迫游泳应激对大鼠情绪和脑细胞外信号调节激酶磷酸化(P-ERK1/2)水平的影响。方法将30只SD雄性大鼠随机分为游泳应激组、装置对照组和空白对照组,每组10只。游泳应激组每天接受5min的游泳应激,装置对照组每天接受5min的新异场景应激,均连续14 d,空白对照组不进行任何干预,然后观察大鼠行为(体质量增长量、旷场测验和糖精水溶液偏好测验)。采用免疫印迹法测定大鼠海马和前额叶皮质的P-ERK1/2。结果(1)游泳应激组在应激7d和应激14d的体质量增长[分别为(75±22)g和(70±24)g]均低于空白对照组[分别为(101±35)g和(115+47)g],均P＜0.05。(2)装置对照组的粪便排泄量[(1.4±1.9)粒]多于空白对照组[(0.4±1.0)粒]和游泳应激组[(0.1±0.3)粒],均P＜0.05；而游泳应激组的水平活动距离[(2077±1245)cm]少于空白对照组[(2990±1038)cm]和装置对照组[(3110±1462)cm],均P＜0.05。(3)游泳应激组的糖精水溶液摄入量[(11±6)g]和糖精水溶液摄入量占总液体摄入量的比例[(37±16)％]均低于空白对照组[分别为(15±4)g和(47±15)％],均P＜0.05。(4)游泳应激组在海马[(46±95)％]和前额叶皮质[(65±24)％]的P-ERK2水平均低于空白对照组[分别为(76±30)%和(99±42)%],均P＜0．05。结论慢性强迫游泳应激能诱发大鼠的抑郁情绪,降低P-ERK2在海马和前额叶皮质的水平。     

异丙酚对小鼠海马脑片细胞外信号调节激酶ERK1/2磷酸化水平的影响

目的 观察异丙酚对小鼠海马脑片细胞外信号调节激酶ERK1／2磷酸化水平的影响。方法BALB／C小鼠，体重18～22g，雌雄不拘，迅速断头取脑，取海马用震动切片机切成450μm厚的脑片。量效实验随机将脑片分为空白对照组（C组）及不同浓度异丙酚组[10^-4mmol／L（P1组）、10^-5mmol／L（P2组）、10^-6mmol／L（P3组）、10^-7mmol／L（P4组）、10^-8mmol／L（P5组）、10^-8mmol／L（P6组）]；分别以不同浓度的异丙酚36℃恒温孵育海马脑片1h。时效实验应用5μmol／L异丙酚孵育脑片，分别于孵育即刻、1、2、5、7、9、12、15、30、60min取出脑片；取5μmol／L异丙酚孵育5min后的部分脑片，以人工脑脊液洗出异丙酚，分别于洗出2、4、7、10、25min取出脑片。应用SDS-PAGE和Westem blot生化技术及Gel Doc凝胶成像系统半定量检测小鼠海马p-ERKI／2水平。结果 异丙酚能降低ERK1／2磷酸化水平，降低呈时间、浓度依赖性（P〈0．05）。随异丙酚孵育5min后洗出时间延长，ERK1／2的磷酸化水平逐渐恢复，但洗出25min时仍明显低于药物处理前水平（P〈0．01）。结论 异丙酚能显著地抑制小鼠海马脑片细胞外信号调节激酶ERK1／2磷酸化水平。     

Responses of CDKs and p53 in Delayed Ischemic Neuronal Death

Stroke is a debilitating disease that affects millions each year.While in many cases cerebral ischemic in jury can be limited by effectivw resuscitation or thrombolytic treatment,the injured neurons wither in a process known as delayed neuronal death(DND).Mounting evidence indicates that DND is not simply necrosis played out in slow motion but apoptosis is triggered.Of particular interest are two groups of signal proteins that participate in apoptosis-cyclin dependent kinases(CDKs) and p53-among a myriad of signaling events after an ischemic insult.Recent investigations have shown that CDKs,a family of enzymes initially known for their role in cell cycle regulation,are activated in injured neurons in DND.As for p53,new reports suggest that its up-regulation may represent a failed attempt to rescue in jured neurons,although its up-regulation was previously considered an indication of apoptosis.These observations thus rekindle an old quest to identify new neuroprotective targets to minimize the stroke damage.In this review,the author will examine the evidence that indicates the participation of CDKs and p53 in DND and then introduce pre-clinical data to explore CDK inhibition as a potential neuroprotective target.Finally,using CDK inhibition as an example,this paper will discuss the pertinent criteria for a viable neuroprotective strategy for ischemic in jury.     

热休克蛋白90对大鼠缺氧心肌细胞丝氨酸苏氨酸蛋白激酶表达的影响

目的探讨内源性热休克蛋白90（HSP90）在缺氧心肌细胞丝氨酸苏氨酸蛋白激酶（AKT）相关信号通路中的作用。方法建立新生Wistar大鼠心肌细胞缺氧模型，将细胞分为正常组、缺氧组、加入HSP90特异性阻断剂格尔德霉素后再缺氧组（格尔德霉素＋缺氧组）。于缺氧后1、3、6、12、24、48h用噻唑蓝法检测心肌细胞的活力；缺氧24h，原位缺口末端标记法检测心肌细胞凋亡指数（AI）；缺氧1、3、6、12、24h，蛋白质印迹法检测大鼠心肌细胞中内源性HSP90及AKT表达水平。结果（1）缺氧24、48h，缺氧组、格尔德霉素＋缺氧组细胞活力均较正常组明显下降（P〈0．05）；格尔德霉素＋缺氧组细胞活力缺氧12h即开始明显下降，缺氧48h时明显低于缺氧组（P〈0．05）。（2）缺氧24h，缺氧组细胞AI为（10．7±1．2）％，明显高于正常组[（1．9±0．3）％．P〈0．05]；格尔德霉素＋缺氧组细胞AI为（26、3±5．3）％，明显高于缺氧组（P〈0．01）。（3）缺氧12h，缺氧组心肌细胞内源性HSP90及AKT表达水平高于正常组与格尔德霉素＋缺氧组；缺氧24h，缺氧组有所下降．格尔德霉素＋缺氧组则下降更明显。结论内源性HSP90对维持心肌细胞的活力有重要作用．缺氧心肌细胞AKT表达水平可受内源性HSP90表达水平的影响。     

17 β雌二醇通过丝裂原活化蛋白激酶抑制前列腺癌细胞系PC3增殖

目的探讨丝裂原活化蛋白激酶在17β雌二醇(E2)抑制前列腺癌PC3细胞生长中的作用。方法检测不同浓度E2作用不同时间后PC3细胞生长抑制率。流式细胞仪分析细胞周期分布,TUNEL染色检测凋亡。Western blot检测ERK1/2,JNK和p38活性。RT-PCR法检测雌激素受体(ER)α、ERβ、P21WAF1和cyclinD1的表达。结果E2抑制PC3细胞增殖,并且可以激活ERK1/2、JNK和p38。处理因素作用48h后,对照组、E2、E2 PD98059、E2 SP600125、E2 SB203580组细胞的凋亡率分别为(0.9±0.1)%;(23.0±1.4)%;(30.0±1.2)%;(10.6±0.8)%和(14.6±0.7)%,(P<0.05)。E2使细胞阻滞在G1期,PD98059、SP600125、SB203580分别预处理1h后细胞分别进一步阻滞在G1期;轻度阻滞在G1期和进入S期。RT-PCR发现PC3细胞表达ERα和ERβ,E2、E2 PD98059、E2 SP600125、E2 SB203580组中cyclinD1、P21WAF1基因表达分别为对照组的(0.42±0.03)、(3.13±0.02)倍;(0.21±0.03)、(3.08±0.05)倍;(0.43±0.01)、(1.31±0.04)倍;(2.81±0.02)、(3.14±0.02)倍(P<0.05)。结论E2激活JNK增加P21WAF1基因表达,并激活p38抑制cyclinD1表达,使细胞阻滞在G1期,JNK和p38通路还介导E2引起PC3细胞凋亡。同时E2又可激活ERK1/2,轻度拮抗上述作用。     

Cannabinoid receptor down-regulation without alteration of the inhibitory effect of CP 55,940 on adenylyl cyclase in the cerebellum of CP 55,940-tolerant mice

The objective of this study was to determine whether the development of tolerance to CP 55,940, a potent cannabinoid agonist, was due to changes in the receptor or second messenger system. ICR mice treated with CP 55,940 (2 mg/kg) twice a day for 6 and one-half days developed a high degree of tolerance to the pharmacological effects of CP 55,940. The ability of CP 55,940 to produce motor hypoactivity, hypothermia and immobility was reduced 163-, 97- and 19-fold, respectively. Evaluation of 3H-CP 55,940 binding to rat brain membranes indicated no difference in receptor affinity between the vehicle- and CP 55,940-treated animals. However, these binding studies revealed a 50% decrease in receptor number in the cerebellum of the CP 55,940-tolerant mice. Although cAMP is generally considered to be the second messenger for cannabinoid receptors, little difference was observed in the inhibitory effects of CP 55,940 on adenylyl cyclase activity in cerebellum between vehicle and drug-treated mice. However, there was an increase in receptor mRNA which suggests a compensation for receptor loss. There are several possible explanations for these results. There may be sufficient spare receptors such that CP 55,940-tolerant mice are capable of producing a maximal effect on the second messenger system. On the other hand, one could conclude that cannabinoid receptor down-regulation does not account for the development of tolerance to all of the effects of CP 55,940 in mice.     

前列腺带性结构及其雄激素、雌激素、表皮生长因子受体分布特征的研究

为了探讨雄激素受体（ＡＲ）、雌激素受体（ＥＲ）、孕激素受体（ＰＲ）和表皮生长因子受体（ＥＧＦＲ）在前列腺带性结构中的分布，应用单饱和剂量测定法，分别测定了前列腺移行带、外周带组织中ＡＲ、ＥＲ、ＰＲ和ＥＧＦＲ的含量。结果表明，在１１例前列腺增生症（ＢＰＨ）患者２２份标本中，移行带和周围带ＡＲ全部阳性；尽管ＡＲ浓度个体差异很大，但是两带之间仍有密切相关性（Ｐ＜０００１）；周围带ＡＲ浓度高于移行带，两带ＡＲ浓度均高于ＰＲ、ＥＲ；ＰＲ和ＥＲ在前列腺带性结构上没有明显差异。ＥＧＦＲ在移行带明显高于周围带。研究证实了ＡＲ、ＥＲ、ＰＲ，ＥＧＦＲ在前列腺移行带和周围带上的分布特征。