安全、便捷技术再生一次性医用口罩的实验研究

在防控新型冠状病毒(2019 novel coronavirus, 2019-nCoV)疫情中,为了减少病毒的传播,一次性医用口罩是普通民众必不可缺少的防护品。然而,随着2019-nCoV的蔓延,口罩短缺现象严重。本研究旨在探讨一次性医用外科口罩(口罩)的再生方法,以达到既有个人防护的效果又能节省资源。用流行性感冒病毒(简称流感)模拟2019-nCoV污染口罩,采用常用的恒温烘箱干烤及电吹风机热风处理2种方法,对表面污染有流感病毒的医用口罩进行病毒灭活,洗脱口罩上已灭活处理的病毒,感染Mardin-Darby狗肾细胞(Mardin-Darby canine kidney cell,MDCK细胞),观察细胞病变并定量检测病毒基因组拷贝数以评价病毒灭活效果。同时采用抽滤系统和PM2.5监测仪对以2种相似热灭活方式处理过的口罩滤过截留PM2.5的效果进行评价。结果显示电吹风机30 min热风处理后病毒基因组拷贝数降低至原来的1/1 000 000～1/10 000 000,接近未感染组,但烘箱56 ℃ 30 min干热处理仅灭活部分病毒。2种热灭活方式对口罩的PM2.5滤过截留效果无显著影响。本研究提供了一个安全、便捷处理一次性医用外科口罩的方法,为个人防护用品口罩表面污染病毒的灭活及其再生利用提供了科学依据。然而,应注意的是在口罩匮乏的非常时期,普通人群可采用该简便技术再生口罩后再次使用,但该方法再生的口罩不适合密切接触患者的人群、医护人员及实验室工作人员使用。     

静脉注射免疫球蛋白制备中的病毒灭活

在静脉注射免疫球蛋白（ＩＶＩＧ）的制备中,采用有机溶剂结合表面活性剂（Ｓ／Ｄ）处理或６０℃１０小时液态加热（巴氏灭活法）的方法对组分Ⅱ（ＩｇＧ）进行病毒灭活处理,灭活效果用指示病毒进行了评价,结果表明：Ｓ／Ｄ法可有效灭活脂包膜病毒ＶＳＶ和Ｓｉｎｄｂｉｓ,巴氏灭活法则对上述病毒以及Ｖａｃｃｉｎｉａ和Ｅｃｈｏ病毒均有较好的灭活作用。经病毒灭活处理的免疫球蛋白,在理化及生物学特性上基本未受到不利影响,用两种方法分别处理组分Ⅱ后制备的ＩＶＩＧ,其主要特性指标均符合该制品的有关质量规定。     

冻干加热处理凝血因子类制剂的病毒灭活验证

在病毒灭活验证过程中,比较了冻干后不同加热方法对凝血因子类制剂中伪狂犬病毒(PRV)的灭活效果,包括80℃干烤72h、100℃水浴加热30min以及100℃蒸汽加热30min方法。结果表明80℃干烤72h对制品中PRV灭活较彻底,另外两种方法对制品中PRV的灭活效果均不理想。     

S／D处理血浆过程中的脂包膜病毒灭活试验观察

通过有机溶剂/去污剂对血浆中指示病毒VSV灭活的观察评估有机溶剂/去污剂对脂包膜病毒死活的效果。血浆样品与VSV病毒按9:1混合,然后用1％TNBP/1％ Triton X-100在30℃处理4h,测定开始样品中的病毒总量和S/D处理后不同时间取样内的病毒总量。实验中样品内加入1％TNBP/1％ Triton X-10015min后VSV病毒已全部灭活,灭活效果≥6.0log。按所述S/D处理方法可以完全灭活血浆内所有的脂包膜病毒而没有主要血浆蛋白的损失。     

利用鸭乙型肝炎病毒感染模型评价血液成分中病毒的灭活效果

目的 利用鸭乙型肝炎病毒(DHBV)感染动物模型,评价亚甲蓝光化学病毒灭活方法对血液成分中DNA病毒的灭活效果。方法 将超离纯化的DHBV分别加入人血浆或人红细胞,经亚甲蓝光化学灭活病毒,将含不同基因组拷贝数DHBV的血浆成分经静脉感染1 d龄雏鸭。采用放射性核素核酸杂交法对血清中DHBV DNA进行检测,计算病毒灭活处理前、后人血浆及人红细胞中DHBV的半数感染计量(ID50)。结果 结果显示加入DHBV的血浆在未经灭活处理前对1 d龄雏鸭的ID50值为103.33,而经病毒灭活处理后ID50值为1010拷贝,灭活处理可使病毒感染性滴度下降达6个Log;加入DHBV的红细胞灭活前ID50值为103.35,经灭活处理后ID50值为108.35拷贝,灭活处理使病毒感染性滴度下降5个Log。结论 利用DHBV感染动物模型,可以检测到少量病毒在自然感染宿主体内的感染性,可用于评判血液成分中病毒灭活方法的效果,亚甲蓝光化学处理对血浆中DNA病毒的灭活效果较好于对红细胞中DNA病毒的灭活作用。     

S／D法处理凝血因子浓缩物类制品的病毒灭活验证

以水泡性口炎病毒 (VSV)为指示病毒 ,验证应用有机溶剂 /去污剂 (简称S/D)法灭活血液制品中病毒的生产工艺 ,并对不同厂家不同批号的四种凝血因子浓缩物类制品 (中间品 )的病毒灭活效果进行了分析总结。结果表明当制品中TNBP和Tween80终浓度为 0 3%和 1 0 % ,在 2 5± 1℃处理 6小时后对于包膜病毒确有显著的灭活效果。     

UV和过硫酸钠联合消毒强化饮用水中微生物的灭活效果

为了研究紫外线联合消毒的协同作用,获得更高效的饮用水不易灭活微生物消毒手段,本研究采用紫外线消毒及过硫酸钠消毒的方式,研究其对大肠杆菌以及枯草芽孢杆菌的灭活效果,通过平板计数法对其灭活效果进行定量评估。结果表明:过硫酸钠协同紫外线消毒,对大肠杆菌灭活有明显效果(紫外光强113.0μW/cm2,过硫酸钠浓度为0.5 mmol/L情况下照射10 min,灭活率超过6 lg);单独过硫酸钠对枯草芽孢杆菌基本无灭活效果,过硫酸钠浓度为0.5 mmol/L情况下10 min的灭活率仅为0.98个对数级;单独紫外线消毒对枯草芽孢杆菌的灭活效果较好,光强为113.0μW/cm2、照射时间10 min可达3.48个对数级;采用联合消毒时,过硫酸钠与紫外线联合消毒在一定程度上对灭活枯草芽孢杆菌有积极影响,最佳情况下可较单独UV消毒和单独过硫酸钠消毒分别增加0.90和3.40个对数级。紫外过硫酸钠联合消毒有望成为一种高效的饮用水微生物消毒方式,并在给水领域得到广泛应用。     

不同消毒方法对家蚕微粒子的存活和侵染力影响

家蚕微粒子病是家蚕Bombyx mori的重要病害,探索其消毒杀灭方法对蚕业生产具有重要意义。本研究采用微粒子染色法和添食家蚕侵染法测试了温度、紫外线和消毒剂处理108个/mL家蚕微粒子后对家蚕微粒子的消杀作用,结果表明：温度对家蚕微粒子虫灭活温度为60℃处理30 min以上;使用20 wx功率紫外灯距离50 cm照射12 min及以上时,微粒子死亡率为47.70%,对家蚕侵染率为0;三氯异氰脲酸800 mg/L及以上浓度处理6 min以上家蚕微粒子死亡率为100%,对家蚕的侵染率为0;戊二醛癸甲溴铵200 mg/L及以上浓度处理6 min以上家蚕微粒子死亡率为100%,对家蚕的侵染率为0。3种消毒方法中温度法主要用于小型蚕具消杀微粒子;紫外线用于蚕业辅助设施的表面微粒子的消杀;三氯异氰脲酸和戊二醛癸甲溴铵均表现出较好的消杀效果,其中戊二醛癸甲溴铵较含氯的消毒剂三氯异氰脲酸稳定性强,刺激性小,对养蚕的金属腐蚀性小,可作为含氯消毒剂的部分替代使用。本研究结果可为蚕业生产中消杀微粒子提供参考。     

过氧化氢干雾对生物安全柜微环境表面消毒效果的研究

生物安全实验室微环境消毒是控制实验室污染的重要环节。过氧化氢广泛用于病原微生物实验室微环境消毒,但其对不同病原微生物的消毒效果有待研究。本文研究了过氧化氢干雾（粒径％10μm）以不同消毒程序对生物安全柜表面常见微生物的消毒效果。结果显示,在生物安全柜内采用优化的消毒程序（发散循环8次,每次1min,达60ppm后,静置消毒2h）,过氧化氢干雾可完全杀灭1×106CFU枯草芽胞杆菌、嗜热脂肪芽胞杆菌、金黄色葡萄球菌、表皮葡萄球菌、耻垢分枝杆菌,以及1×106CFU大肠埃希菌。然而,当金黄色葡萄球菌、表皮葡萄球菌、耻垢分枝杆菌浓度达1×107CFU时,过氧化氢干雾无法完全杀灭。因此,建议在进行过氧化氢干雾消毒时,应先用消毒剂处理,以期彻底杀灭生物安全柜微环境中污染的病原微生物。     

亚甲兰光化学法对血浆乙肝病毒的灭活

目的:研究灭活病毒对乙肝病毒和血浆成分的影响。方法:在已知HBV-DNA阳性血浆中加入MB,调整MB终浓度分别为1.0μmol/l、5.0μmol/l、10.0μmol/l,光照度为40000LUX,照射时间分别为0min、20min、40min、60min,在各点分别取样抽提基因组DNA用于荧光定量分析,测定HBV-DNA的浓度,推断在不同时间和浓度下的灭活效果。在灭活效果最好的点取样检测血浆正常成分有无显著变化,其中包括生化指标,酶学指标,凝血因子FⅧ:C活性,SDS聚丙烯酰胺凝胶电泳观察蛋白亚基的变化,蛋白免疫印迹观察血浆的抗体蛋白活性变化。结果:当MB为10.0μmol/L,光照60min时,灭活乙肝病毒的效果最好,对于高拷贝数的病毒浓度,在本实验的条件下,尚无法完全灭活病毒,但可以使HBV-DNA浓度明显下降;从病毒灭活的表格看,灭活效果与时间和MB浓度呈正相关,作用60min时血浆中主要成分的生化指标无显著变化及凝血因子FⅧ:C活性无明显改变,部分血浆酶活性有明显下降(P<0.05)。血浆蛋白的亚基数目和迁移速度未见明显变化,血浆的抗体蛋白也具有正常的生物学活性。结论:MB10μmol/L辅助荧光...     

State-dependent inactivation of the alpha1G T-type calcium channel.

We have examined the kinetics of whole-cell T-current in HEK 293 cells stably expressing the alpha1G channel, with symmetrical Na(+)(i) and Na(+)(o) and 2 mM Ca(2+)(o). After brief strong depolarization to activate the channels (2 ms at +60 mV; holding potential -100 mV), currents relaxed exponentially at all voltages. The time constant of the relaxation was exponentially voltage dependent from -120 to -70 mV (e-fold for 31 mV; tau = 2.5 ms at -100 mV), but tau = 12-17 ms from-40 to +60 mV. This suggests a mixture of voltage-dependent deactivation (dominating at very negative voltages) and nearly voltage-independent inactivation. Inactivation measured by test pulses following that protocol was consistent with open-state inactivation. During depolarizations lasting 100-300 ms, inactivation was strong but incomplete (approximately 98%). Inactivation was also produced by long, weak depolarizations (tau = 220 ms at -80 mV; V(1/2) = -82 mV), which could not be explained by voltage-independent inactivation exclusively from the open state. Recovery from inactivation was exponential and fast (tau = 85 ms at -100 mV), but weakly voltage dependent. Recovery was similar after 60-ms steps to -20 mV or 600-ms steps to -70 mV, suggesting rapid equilibration of open- and closed-state inactivation. There was little current at -100 mV during recovery from inactivation, consistent with 相似文献   

Dynamical characterization of inactivation path in voltage-gated Na+ ion channel by non-equilibrium response spectroscopy

Inactivation path of voltage gated sodium channel has been studied here under various voltage protocols as it is the main governing factor for the periodic occurrence and shape of the action potential. These voltage protocols actually serve as non-equilibrium response spectroscopic tools to study the ion channel in non-equilibrium environment. In contrast to a lot of effort in finding the crystal structure based molecular mechanism of closed-state(CSI) and open-state inactivation(OSI); here our approach is to understand the dynamical characterization of inactivation. The kinetic flux as well as energetic contribution of the closed and open- state inactivation path is compared here for voltage protocols, namely constant, pulsed and oscillating. The non-equilibrium thermodynamic quantities used in response to these voltage protocols serve as improved characterization tools for theoretical understanding which not only agrees with the previously known kinetic measurements but also predict the energetically optimum processes to sustain the auto-regulatory mechanism of action potential and the consequent inactivation steps needed. The time dependent voltage pattern governs the population of the conformational states which when couple with characteristic rate parameters, the CSI and OSI selectivity arise dynamically to control the inactivation path. Using constant, pulsed and continuous oscillating voltage protocols we have shown that during depolarization the OSI path is more favored path of inactivation however, in the hyper-polarized situation the CSI is favored. It is also shown that the re-factorisation of inactivated sodium channel to resting state occurs via CSI path. Here we have shown how the subtle energetic and entropic cost due to the change in the depolarization magnitude determines the optimum path of inactivation. It is shown that an efficient CSI and OSI dynamical profile in principle can characterize the open-state drug blocking phenomena.     

Shab K+ channel slow inactivation

Herein, we report the first characterization of Shab slow inactivation. Open Shab channels inactivate within seconds, with two voltage-independent time constants. Additionally, Shab presents significant closed-state inactivation. We found that with short depolarizing pulses, shorter than the slowest inactivation time constant, the resulting inactivation curve has a marked U-shape, but as pulse duration increases, approaching steady-state conditions, the U-shape vanishes, and the resulting inactivation curves converge to the classical Boltzmann h_∞ curve. Regarding the mechanism of inactivation, we found that external K⁺ and TEA facilitate both open- and closed-state inactivation, while the cavity blocker quinidine hinders inactivation. These results together with our previous observations regarding the K⁺-dependent stability of the K⁺ conductance, suggest the novel hypothesis that inactivation of Shab channels, and possibly that of other Kv channels whose inactivation is facilitated by K⁺, does not involve a significant narrowing of the extracellular entry of the pore. Instead, we hypothesize that there is only a rearrangement of a more internal segment of the pore that affects the central cavity and halts K⁺ conduction.     

Evaluation of microfluidics reactor technology on the kinetics of virus inactivation

Mammalian cell lines constitute an important part in the manufacture of therapeutic proteins. However, their susceptibility to virus contamination is a potential risk to patient safety and productivity, and has led to the development of a repertoire of virus inactivation techniques. From a process development viewpoint, the challenge is to demonstrate the required log reduction in virus content without a significant loss in product titer or quality. The balance between the two is dictated by the kinetics of virus inactivation and protein degradation, both of which are critically affected by process parameters. In this study we describe a commercially available microchannel reactor (MCR) and demonstrate how it can be used to evaluate the impact of temperature on the kinetics of virus inactivation and protein product degradation. Virus spiking experiments are reported using Xenotropic Murine Leukemia Virus and REOvirus, into buffers in the absence and presence of a therapeutic protein currently under development at Lilly. The results demonstrate that the MCR is an ideal platform for evaluation of fast reactive systems and reactions that are particularly sensitive to small changes to process conditions. These conditions include heat inactivation of a virus in a mammalian cell culture process stream used in the manufacture of therapeutic proteins and antibodies.     

哺乳动物X染色体失活机制

哺乳动物X染色体连锁基因的剂量平衡,是通过雌性胚胎发育早期随机或印记失活一条X染色体来实现的,这是一个复杂的过程,包括：启动、计数、选择、维持等一系列的步骤。X染色体失活中心是X染色体失活的主控开关座位,调节X失活的早期事件,失活发生后,X染色体的失活状态可稳定地存在并传递给后代,这一过程涉及基因组印记的形成。此外,在雄性动物,精原细胞减数分裂早期也存在着短暂的X染色体失活现象。现对哺乳动物X染色体失活机制的最新进展进行综述。     

The Effect of Urea on Acid Phosphatase Deactivation

Acid phosphatase thermal deactivation follows a complex path consisting of an initial decay of the native enzyme towards an equilibrium distribution of two intermediate structures, mutually at equilibrium. This initial transition is followed by a final decay towards a completely inactive enzyme configuration.

All the relevant parameters (one equilibrium and two kinetic constants) of the phenomenon are environment-sensitive. It is shown that urea affects the deactivation, by increasing the rate of both structural transitions as well as the thermodynamics of the equilibrium between intermediate forms. For every urea concentration up to 2.4M, an equivalent temperature can be calculated that yields exactly the same activity versus time profile. The result suggests that enzyme deactivation is controlled by a single parameter. Entirely different environments, so long as they result in the same value of the latter, are therefore bound to produce the same deactivation profile.

Marked deviations from thermal equivalence become apparent at higher urea concentrations. Therefore, extremely high urea concentrations seems to give rise to a change in the deactivation mechanism.     

Sterilization of ginseng using a high pressure CO2 at moderate temperatures

The aim of this study was to determine the feasibility of using high pressure CO2 for sterilization of Ginseng powder, as an alternative method to conventional techniques such as gamma-irradiation and ethylene oxide. The Ginseng sample used in this study was originally contaminated with fungi and 5 x 10(7) bacteria/g that was not suitable for oral use. This is the first time that high pressure CO2 has been used for the sterilization of herbal medicine to decrease the total aerobic microbial count (TAMC) and fungi. The effect of the process duration, operating pressure, temperature, and amount of additives on the sterilization efficiency of high pressure CO2 were investigated. The process duration was varied over 15 h; the pressure between 100 and 200 bar and the temperature between 25 and 75 degrees C. A 2.67-log reduction of bacteria in the Ginseng sample was achieved after long treatment time of 15 h at 60 degrees C and 100 bar, when using neat carbon dioxide. However, the addition of a small quantity of water/ethanol/H2O2 mixture, as low as 0.02 mL of each additive/g Ginseng powder, was sufficient for complete inactivation of fungi within 6 h at 60 degrees C and 100 bar. At these conditions the bacterial count was decreased from 5 x 10(7) to 2.0 x 10(3) TAMC/g complying with the TGA standard for orally ingested products. A 4.3 log reduction in bacteria was achieved at 150 bar and 30 degrees C, decreasing the TAMC in Ginseng sample to 2,000, below the allowable limit. However, fungi still remained in the sample. The complete inactivation of both bacteria and fungi was achieved within 2 h at 30 degrees C and 170 bar using 0.1 mL of each additive/g Ginseng. Microbial inactivation at this low temperature opens an avenue for the sterilization of many thermally labile pharmaceutical and food products that may involve sensitive compounds to gamma-radiation and chemically reactive antiseptic agents.     

Effect of Additives on the Inactivation of Lysozyme Mediated by Free Radicals Produced in the Thermolysis of 2, 2-Azo-Bis-(2-Amidinopropane)

The inactivation of lysozyme caused by the radicals produced by thermolysis of 2, 2-azo-bis-2-amidino-propane can be prevented by the addition of different compounds that can react with the damaging free radicals. Compounds of high reactivity (propyl gallate, Trolox, cysteine, albumin, ascorbate, and NADH) afford almost total protection until their consumption, resulting in well-defined induction times. The number of radicals trapped by each additive molecule consumed ranges from 3 (propyl gallate) to 0.12 (cysteine). This last value is indicative of chain oxidation of the inhibitor. Uric acid is able to trap nearly 2.2 radicals per added molecule, but even at large (200 μM) concentrations, a residual inactivation of the enzyme is observed, which may be caused by urate-derived radicals.

Compounds of lower reactivity (tryptophan, Tempol, hydroquinone, desferrioxamine, diethylhydroxylamine, methionine, histidine, NAD⁺ and tyrosine) only partially decrease the lysozyme inactivation rates. For these compounds, we calculated the concentration necessary to reduce the enzyme inactivation rate to one half of that observed in the absence of additives. These concentrations range from 9 μM (tryptophan and Tempol) to 5 mM (NAD⁺).     

Efficient protein depletion by genetically controlled deprotection of a dormant N‐degron

Methods that allow for the manipulation of genes or their products have been highly fruitful for biomedical research. Here, we describe a method that allows the control of protein abundance by a genetically encoded regulatory system. We developed a dormant N‐degron that can be attached to the N‐terminus of a protein of interest. Upon expression of a site‐specific protease, the dormant N‐degron becomes deprotected. The N‐degron then targets itself and the attached protein for rapid proteasomal degradation through the N‐end rule pathway. We use an optimized tobacco etch virus (TEV) protease variant combined with selective target binding to achieve complete and rapid deprotection of the N‐degron‐tagged proteins. This method, termed TEV protease induced protein inactivation (TIPI) of TIPI‐degron (TDeg) modified target proteins is fast, reversible, and applicable to a broad range of proteins. TIPI of yeast proteins essential for vegetative growth causes phenotypes that are close to deletion mutants. The features of the TIPI system make it a versatile tool to study protein function in eukaryotes and to create new modules for synthetic or systems biology.     

Effect of Additives on the Inactivation of Lysozyme Mediated by Free Radicals Produced in the Thermolysis of 2, 2-Azo-Bis-(2-Amidinopropane)

The inactivation of lysozyme caused by the radicals produced by thermolysis of 2, 2-azo-bis-2-amidino-propane can be prevented by the addition of different compounds that can react with the damaging free radicals. Compounds of high reactivity (propyl gallate, Trolox, cysteine, albumin, ascorbate, and NADH) afford almost total protection until their consumption, resulting in well-defined induction times. The number of radicals trapped by each additive molecule consumed ranges from 3 (propyl gallate) to 0.12 (cysteine). This last value is indicative of chain oxidation of the inhibitor. Uric acid is able to trap nearly 2.2 radicals per added molecule, but even at large (200 μM) concentrations, a residual inactivation of the enzyme is observed, which may be caused by urate-derived radicals.

Compounds of lower reactivity (tryptophan, Tempol, hydroquinone, desferrioxamine, diethylhydroxylamine, methionine, histidine, NAD⁺ and tyrosine) only partially decrease the lysozyme inactivation rates. For these compounds, we calculated the concentration necessary to reduce the enzyme inactivation rate to one half of that observed in the absence of additives. These concentrations range from 9 μM (tryptophan and Tempol) to 5 mM (NAD⁺).