Physical Methods of Disinfection (A Review)

Plasma Physics Reports - The basic physical methods of disinfection of air, water, and surfaces, such as filtration, ozonation, exposure to ultraviolet radiation, photocatalysis, cold plasma,...     

次氯酸钠和洗必泰作为牙根管消毒冲洗液对根管内的感染物质的影响

摘要 目的：探究次氯酸钠和洗必泰作为牙根管消毒冲洗液的对根管内感染物质的影响。方法：选取76例接受根管治疗的患者根据随机数字表法分为观察组和对照组2组,其中对照组患者采用次氯酸钠治疗,观察组患者则采用次氯酸钠和洗必泰联合治疗。对比分析两组患者根管内细菌菌落计数、根管内感染物质、治疗效果、远期有效率和不良反应发生率。结果：根管消毒冲洗后观察组根管内细菌菌落计数和根管内感染物质显著低于对照组,差异具有统计学意义（P<0.05）;观察组患者治疗总有效率和不良反应的发生率分别为100.00%和13.16%,对照组患者治疗总有效率和不良反应的发生率分别为88.00%和65.79%,差异具有统计学意义（P<0.05）。另外,观察组患者第3个月、第6个月和1年后的治疗有效率均大于对照组。结论：次氯酸钠和洗必泰作为牙根管消毒冲洗液能够有效的减少根管内的感染物质的数量,提高治疗有效率,降低不良反应的发生率,值得临床推广使用。     

木薯外植体快速、高效消毒的简易方法

本研究以木薯栽培种华南7号的幼嫩茎段为外植体,就取材地点,取材时间,取材天气,消毒试剂进行筛选研究,以发展一套木薯外植体快速、有效消毒的简易方法.实验结果表明,来自室内培养植株的外植体接种污染率低.大田取样中午脱菌效果好于清晨和傍晚.连续晴天取材脱菌效果比雨后第2天稍好.0.1%HgCl2对室内所取材料消毒效果好于10%NaClO的消毒效果,前者无菌率平均高达90%以上.     

4-(O-Benzylphenoxy)-N-Methylbutylamine (Bifemelane) and Other 4-(O-Benzylphenoxy)-N-Methylalkylamines as New Inhibitors of Type A and B Monoamine Oxidase

4-(O-Benzylphenoxy)-N-methylbutylamine (Bifemelane, BP-N-methylbutylamine), a new psychotropic drug, was found to inhibit monoamine oxidase (MAO) in human brain synaptosomes. It inhibited type A MAO (MAO-A) competitively and type B (MAO-B) noncompetitively. BP-N-methylbutylamine had a much higher affinity to MAO-A than an amine substrate, kynuramine, and it was a more potent inhibitor of MAO-A than of MAO-B. The Ki values of MAO-A and -B were determined to be 4.20 and 46.0 microM, respectively, while the Km values of MAO-A and -B with kynuramine were 44.1 and 90.0 microM, respectively. The inhibition of MAO-A and -B by BP-N-methylbutylamine was found to be reversible by dialysis of the incubation mixture. MAO-A in human placental and liver mitochondria and in a rat clonal pheochromocytoma cell line, PC12h, was inhibited competitively by BP-N-methylbutylamine, while MAO-B in human liver mitochondria was inhibited noncompetitively, as in human brain synaptosomes. BP-N-methylbutylamine was not oxidized by MAO-A and -B. The effects of other BP-N-methylalkylamines, such as BP-N-methylethylamine, -propylamine, and -pentanylamine, on MAO activity were examined. BP-N-methylbutylamine was the most potent inhibitor of MAO-A, and BP-N-methylethylamine and -propylamine inhibited MAO-B competitively, whereas BP-N-methylbutylamine and -pentanylamine inhibited it noncompetitively. Inhibition of these BP-N-methylalkylamines on MAO-A and -B is discussed in relation to their chemical structure.     

Amplification of Nucleic Acids by Polymerase Chain Reaction (PCR) and Other Methods and their Applications

Abstract

The in vitro replication of DNA, principally using the polymerase chain reaction (PCR), permits the amplification of defined sequences of DNA. By exponentially amplifying a target sequence, PCR significantly enhances the probability of detecting target gene sequences in complex mixtures of DNA. It also facilitates the cloning and sequencing of genes. Amplification of DNA by PCR and other newly developed methods has been applied in many areas of biological research, including molecular biology, biotechnology, and medicine, permitting studies that were not possible before. Nucleic acid amplification has added a new and revolutionary dimension to molecular biology. This review examines PCR and other in vitro nucleic acid amplification methodologies—examining the critical parameters and variations and their widespread applications—giving the strengths and limitations of these methodologies.     

Solution structure of the toluene 4-monooxygenase effector protein (T4moD)

Toluene 4-monooxygenase (T4MO) from Pseudomonas mendocina catalyzes the NADH- and O(2)-dependent hydroxylation of toluene to form p-cresol. The complex consists of an NADH oxidoreductase (T4moF), a Rieske ferredoxin (T4moC), a diiron hydroxylase [T4moH, with (alphabetagamma)(2) quaternary structure], and a catalytic effector protein (T4moD). The solution structure of the 102-amino acid T4moD effector protein has been determined from 2D and 3D (1)H, (13)C, and (15)N NMR spectroscopic data. The structural model was refined through simulated annealing by molecular dynamics in torsion angle space (DYANA software) with input from 1467 experimental constraints, comprising 1259 distance constraints obtained from NOEs, 128 dihedral angle constraints from J-couplings, and 80 hydrogen bond constraints. Of 60 conformers that met the acceptance criteria, the 20 that best satisfied the input constraints were selected to represent the solution structure. With exclusion of the ill-defined N- and C-terminal segments (Ser1-Asn11 and Asp99-Met102), the atomic root-mean-square deviation for the 20 conformers with respect to the mean coordinates was 0.71 A for the backbone and 1.24 A for all non-hydrogen atoms. The secondary structure of T4moD consists of three alpha-helices and seven beta-strands arranged in an N-terminal betaalphabetabeta and a C-terminal betaalphaalphabetabetabeta domain topology. Although the published NMR structures of the methane monooxygenase effector proteins from Methylosinus trichosporium OB3b and Methylococcus capsulatus (Bath) have a similar secondary structure topology, their three-dimensional structures differ from that of T4moD. The major differences in the structures of the three effector proteins are in the relative orientations of the two beta-sheets and the interactions between the alpha-helices in the two domains. The structure of T4moD is closer to that of the methane monooxygenase effector protein from M. capsulatus (Bath) than that from M. trichosporium OB3b. The specificity of T4moD as an effector protein was investigated by replacing it in reconstituted T4MO complexes with effector proteins from monooxygenases from other bacterial species: Pseudomonas pickettii PKO1 (TbuV, toluene 3-monooxygenase); Pseudomonas species JS150 (TbmC, toluene 2-monooxygenase); and Burkeholderia cepacia G4 (S1, toluene 2-monooxygenase). The results showed that the closely related TbuV effector protein (55% sequence identity) provided partial activation of the complex, whereas the more distantly related TbmC (34% sequence identity) and S1 (29% sequence identity) did not. The (1)H NMR chemical shifts of the side-chain amide protons of Asn34, a conserved, structurally relevant amino acid, were found to be similar in spectra of effector proteins T4moD and TbuV but not in the spectrum of TbmC. This suggests that the region around Asn34 may be involved in structural aspects contributing to functional specificity.     

Photoregulation of Phosphoenolpyruvate Carboxylase in Salsola soda L. and Other C(4) Plants

Photoactivation of phosphoenolpyruvate carboxylase was found to occur in several, though not all, C₄ species examined; Salsola soda L. was used for a detailed study of this effect of light.

Activity differences between light and darkness are maximized when glycerol (25% v/v) is included in the extraction medium and in the absence of mercaptoethanol. In plants grown in the growth chamber, the night-form of the enzyme, in addition to low activity, shows a positive cooperativity (with phosphoenolpyruvate), which is gradually abolished by light of increasing intensities. This allosteric behavior is absent in plants adapted to a high light environment. Activation and deactivation, under light and darkness respectively, are quite fast, suggesting post-translational regulation. The photoactivation appears to depend on photosynthetic electron flow, since it is saturated at high photon fluxes (around 1000 microeinsteins per square meter per second) and inhibited by 3-(3,4-dichlorophenyl)-1,1-dimethylurea.

     

Proteomic Profiling of Detergent Resistant Membranes (Lipid Rafts) of Prostasomes

Prostasomes are exosomes derived from prostate epithelial cells through exocytosis by multivesicular bodies. Prostasomes have a bilayered membrane and readily interact with sperm. The membrane lipid composition is unusual with a high contribution of sphingomyelin at the expense of phosphatidylcholine and saturated and monounsaturated fatty acids are dominant. Lipid rafts are liquid-ordered domains that are more tightly packed than the surrounding nonraft phase of the bilayer. Lipid rafts are proposed to be highly dynamic, submicroscopic assemblies that float freely within the liquid disordered membrane bilayer and some proteins preferentially partition into the ordered raft domains. We asked the question whether lipid rafts do exist in prostasomes and, if so, which proteins might be associated with them. Prostasomes of density range 1.13–1.19g/ml were subjected to density gradient ultracentrifugation in sucrose fabricated by phosphate buffered saline (PBS) containing 1% Triton X-100 with capacity for banding at 1.10 g/ml, i.e. the classical density of lipid rafts. Prepared prostasomal lipid rafts (by gradient ultracentrifugation) were analyzed by mass spectrometry. The clearly visible band on top of 1.10g/ml sucrose in the Triton X-100 containing gradient was subjected to liquid chromatography-tandem MS and more than 370 lipid raft associated proteins were identified. Several of them were involved in intraluminal vesicle formation, e.g. tetraspanins, ESCRTs, and Ras-related proteins. This is the first comprehensive liquid chromatography-tandem MS profiling of proteins in lipid rafts derived from exosomes. Data are available via ProteomeXchange with identifier PXD002163.Extracellular vesicles (EVs)¹ are membrane surrounded structures that exist in all body fluids and all cells studied so far release EVs (1). They are heterogeneous, spherical organelles spanning between 30 to more than 1000 nm in diameter and include exosomes, microvesicles, and apoptotic bodies (2). There is increasing evidence supporting the important role of EVs in cell-to-cell communication by their delivery of proteins, lipids, and nucleic acids from one donor cell to many target cells. The generation of exosomes/prostasomes is a complicated process involving two invagination sessions of biological membranes. The first one comprises the plasma membrane contributing with endocytic vesicles in the formation of early endosomes that mature into late endosomes. The second one starts multiple inward buddings of the late endosomal membrane creating intraluminal vesicles (ILVs) therewith completing formation of multivesicular bodies (MVBs) or storage vesicles (3) thus retaining selected molecules from the maternal cell. Ceramide can induce such formation of small microdomains into larger domains (4). Ceramide is one of two cleavage products of sphingomyelin by sphingomyelinase, the other is phosphocholine (5) and prostasomes contain sphingomyelinase (6). The membrane of MVBs (storage vesicles) may fuse with the plasma membrane of the secretory cell and, in case of prostate epithelial cells, release the intraluminal vesicles as prostasomes to the extracellular space (7, 8). It is noteworthy that the bilayered membrane surrounding prostasomes (after the two sessions of invaginations) should be regarded as “right-side-out” with reference to the plasma membrane. This is illustrated by e.g. Mg²⁺ and Ca²⁺ -stimulated ATPase that is an ectoenzyme (9) that is also appearing at the outer surface of prostasomes (10). The corollary is that cell surface interactive molecules like enzymes and receptors may appear also on the membranes of exosomes/prostasomes.The majority of prostasomes ranges in diameter-size from 30–200 nm, with a mean of 142 nm (11). The main purpose of prostasomes may be to transfer newly synthesized proteins from the prostate gland to sperm and thereby, among other things, render them protection in the female genital tract (12, 13). Prostasomal proteins may be transferred to sperm through different mechanisms, viz direct interaction with the sperm membrane (14), fusion at a lowered pH (15), and internalization (16). Prostasomes are immunosuppressive and regulate the complement system and they have proven antioxidant and antibacterial properties (17, 18). Prostasomes contain a surrounding lipid membrane bilayer that exhibits a high cholesterol/phospholipid ratio (19). The lipid composition of the membrane is unusual and among the phospholipids sphingomyelin is the dominant one, contrary to other cell membranes where phosphatidylcholine is most abundant. Prostasomes have a strong contribution of saturated and monounsaturated fatty acids (19, 20). These characteristics together with a high cholesterol/phospholipid ratio make the membrane of the prostasome very stable as demonstrated by electron spin resonance (19).In the early 1970s the plasma membrane of the cell was described as a fluid mosaic by Singer and Nicholson (21), but as early as in 1953 Palade claimed that in the bilayered lipid membrane, proposed by Davson and Danielli (22), were areas of different composition, so called caveolae (23). These caveolae are invaginations of the plasma membrane (24). The first hypothesis of lipid rafts (specialized membrane domains enriched in glycosphingolipids, proteins and cholesterol) was brought up in 1988 by van Meer and Simons (25) and was subsequently elaborated in 1997 by Simons and Ikonen (26). Lipid rafts were defined as low density subdomains of the plasma membrane that are resistant to nonionic detergents at a low temperature (27, 28). Fatty acids present in lipid rafts are more saturated, compared with the membrane adjacent to the domains. It means that the fatty acids can be packed more densely and this may lead to phase separation. The abundance of intercalating cholesterol makes the rafts more rigid and less fluid than the rest of the plasma membrane (29). In other words, the membrane can undergo phase separation into co-existing liquid-disordered and liquid-ordered phases. The liquid-ordered phase (the lipid raft) becomes enriched in cholesterol and saturated fatty acids and is characterized by tight lipid packing and reduced molecular diffusion, as we noticed for prostasomes (19).There are two different types of lipid rafts, planar and caveolae. The distinguishing factor is that the caveolae are formed by the protein caveolin whereas the planar rafts lack this protein (30). Instead they contain the protein flotillin (31). Researchers have found that selected proteins localize, and colocalize in lipid rafts (32). Lipid rafts are not anchored at a specific site in the plasma membrane, but float freely. This enables larger and more stable platform domains to aggregate (33). The formed aggregates are involved in many biological functions including endocytosis, cell communication, molecular trafficking, neurotransmission and they could be understood as organizing centers for signaling molecules and receptors (30, 31). When cells are depleted of cholesterol, e.g. by the agent methyl-β-cyclodextrin, formation of caveolae expression and also raft-dependent endocytosis are inhibited (34). This demonstrates the importance of these cholesterol-enriched domains to cell survival. Flotillins are also involved in endocytosis in a process controlled by the phosphorylation of tyrosine residues (35).In this work we asked the question whether lipid rafts do exist in prostasomes and, if so, which proteins might be associated with them. Accordingly, we prepared lipid rafts from human prostasomes in order to characterize their protein content.     

Use of RAPD PCR to Distinguish the B Biotype from Other Biotypes of Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae)

The use of RAPD PCR to identify the B biotype of the whitefly Bemisia tabaci and distinguish it from other biotypes and species of whitefly is described. the technique enables the use of alcohol preserved material instead of live or frozen material as required by allozyme electrophoresis and demonstrates that eggs, juvenile stages and males or females can all be used.     

Toxicity of an Anionic Detergent to the Spawn and Larvae of Anurans (Amphibia)

Several experiments were performed to test the toxicity of FIT, a washing-up and general cleaning liquid containing about 10 % sodiumalkanemonosulfonate (industrial name E 30) to spawn and larvae of anura. The LC₅₀ values for spawn were between 1.62 × 10⁻³ % (Rana dalmatina) and 4.97 × 10⁻³ % (Rana temporaria). The sensitivity of anuran larvae depends on the concentration and duration of exposure to the detergent as well as on the developmental and differentiation stage of the organisms. The toxicity of FIT is not due solely to the alkanesulfonate it contains, but results from the joint action of the surfactant, polyphosphates and solvents. The results indicate that detergents may be harmful to the amphibian fauna of Central Europe.     

Efficacy of Conventional Endoscopic Disinfection and Sterilization Methods Against Helicobacter pylori Contamination

Background. Iatrogenic transmission of Helicobacter pylori via contaminated endoscopic devices is well documented. Despite the prevalence of this infectious agent, few controlled studies have investigated the major factors that impact on reprocessing of endoscopes contaminated with H. pylori. Materials and Methods. An endoscope (Pentax) was contaminated with 10⁸ cfu/ml of H. pylori in 5% bovine calf serum as standardized inoculum. The endoscope then was passed through one of eight arms (five repetitions per arm = 40 total runs), as follows: 1, recovery control (no cleaning or disinfection); 2, manual cleaning alone; 3–5, manual precleaning followed by either 10-, 20-, or 45-minute exposure to 2% glutaraldehyde and ethanol (ETOH) drying; 6, manual cleaning followed by automated reprocessing by STERIS System; 7 and 8, automated reprocessing by STERIS with and without active peracetic acid sterilant (wash-off control). Suction-biopsy channels and air-water channels were harvested for microbiological culture. Results. Control runs recovered more than 1 × 10⁶ cfu per site, confirming the viability of the test organism and the adequacy of the biological burden for challenge. When instruments underwent manual cleaning alone (without subsequent disinfection), test organisms remained in 40% of runs at the air-water site. Manual cleaning followed by 10-, 20-, or 45-minute glutaraldehyde exposure and ETOH drying removed all test organisms from all sites in all runs (i.e., 100% disinfection). The automated STERIS system with or without active peracetic acid sterilant also removed all test organisms from all sites in all runs, as did manual cleaning followed by STERIS use. Conclusion. Manual cleaning alone does not effectively remove H. pylori from an endoscope. Current joint association recommendations for minimal disinfection (manual cleaning followed by at least 20 minutes of immersion in glutaraldehyde and ETOH drying) are effective in preventing cross-transmission of H. pylori. Reprocessing using the automated STERIS system according to manufacturer's recommendations also is highly effective in sterilizing endoscopes contaminated with H. pylori.     

Detergent Screening and Purification of the Human Liver ABC Transporters BSEP (ABCB11) and MDR3 (ABCB4) Expressed in the Yeast Pichia pastoris

The human liver ATP-binding cassette (ABC) transporters bile salt export pump (BSEP/ABCB11) and the multidrug resistance protein 3 (MDR3/ABCB4) fulfill the translocation of bile salts and phosphatidylcholine across the apical membrane of hepatocytes. In concert with ABCG5/G8, these two transporters are responsible for the formation of bile and mutations within these transporters can lead to severe hereditary diseases. In this study, we report the heterologous overexpression and purification of human BSEP and MDR3 as well as the expression of the corresponding C-terminal GFP-fusion proteins in the yeast Pichia pastoris. Confocal laser scanning microscopy revealed that BSEP-GFP and MDR3-GFP are localized in the plasma membrane of P. pastoris. Furthermore, we demonstrate the first purification of human BSEP and MDR3 yielding ∼1 mg and ∼6 mg per 100 g of wet cell weight, respectively. By screening over 100 detergents using a dot blot technique, we found that only zwitterionic, lipid-like detergents such as Fos-cholines or Cyclofos were able to extract both transporters in sufficient amounts for subsequent functional analysis. For MDR3, fluorescence-detection size exclusion chromatography (FSEC) screens revealed that increasing the acyl chain length of Fos-Cholines improved monodispersity. BSEP purified in n-dodecyl-β-D-maltoside or Cymal-5 after solubilization with Fos-choline 16 from P. pastoris membranes showed binding to ATP-agarose. Furthermore, detergent-solubilized and purified MDR3 showed a substrate-inducible ATPase activity upon addition of phosphatidylcholine lipids. These results form the basis for further biochemical analysis of human BSEP and MDR3 to elucidate the function of these clinically relevant ABC transporters.     

A New Artificial Chaperone for Protein Refolding: Sequential Use of Detergent and Alginate

A novel artificial chaperone system using a combination of detergents and alginate was developed to refold three enzymes with totally different structures. Upon dilution of denatured protein in the presence of the capturing agent, complexes of the detergent and non-native protein molecules are formed and thereby the formation of protein aggregates is prevented. The so-called captured protein is unable to refold from the detergent-protein complex states unless a stripping agent is used to gradually remove the detergent molecules. In that respect, we used alginate, a linear copolymer of d-mannuronic acid and l-guluronic acid, to initiate and complete the refolding process. The results indicated that the extent of refolding assistance for the proteins was different due to detergent structure and also the length of hydrophobic portion of each detergent. These observed differences were attributed to the strong electrostatic and hydrophobic interactions among the capturing and stripping agents used in this investigation. Based on this newly developed method, it is expected that the protein refolding operation can be achieved easily, cheaply and efficiently.     

The Use of Amphipols for Solution NMR Studies of Membrane Proteins: Advantages and Constraints as Compared to Other Solubilizing Media

Solution-state nuclear magnetic resonance studies of membrane proteins are facilitated by the increased stability that trapping with amphipols confers to most of them as compared to detergent solutions. They have yielded information on the state of folding of the proteins, their areas of contact with the polymer, their dynamics, water accessibility, and the structure of protein-bound ligands. They benefit from the diversification of amphipol chemical structures and the availability of deuterated amphipols. The advantages and constraints of working with amphipols are discussed and compared to those associated with other non-conventional environments, such as bicelles and nanodiscs.     

Stability of the Neurotensin Receptor NTS1 Free in Detergent Solution and Immobilized to Affinity Resin

Background

Purification of recombinant membrane receptors is commonly achieved by use of an affinity tag followed by an additional chromatography step if required. This second step may exploit specific receptor properties such as ligand binding. However, the effects of multiple purification steps on protein yield and integrity are often poorly documented. We have previously reported a robust two-step purification procedure for the recombinant rat neurotensin receptor NTS1 to give milligram quantities of functional receptor protein. First, histidine-tagged receptors are enriched by immobilized metal affinity chromatography using Ni-NTA resin. Second, remaining contaminants in the Ni-NTA column eluate are removed by use of a subsequent neurotensin column yielding pure NTS1. Whilst the neurotensin column eluate contained functional receptor protein, we observed in the neurotensin column flow-through misfolded NTS1.

Methods and Findings

To investigate the origin of the misfolded receptors, we estimated the amount of functional and misfolded NTS1 at each purification step by radio-ligand binding, densitometry of Coomassie stained SDS-gels, and protein content determination. First, we observed that correctly folded NTS1 suffers damage by exposure to detergent and various buffer compositions as seen by the loss of [³H]neurotensin binding over time. Second, exposure to the neurotensin affinity resin generated additional misfolded receptor protein.

Conclusion

Our data point towards two ways by which misfolded NTS1 may be generated: Damage by exposure to buffer components and by close contact of the receptor to the neurotensin affinity resin. Because NTS1 in detergent solution is stabilized by neurotensin, we speculate that the occurrence of aggregated receptor after contact with the neurotensin resin is the consequence of perturbations in the detergent belt surrounding the NTS1 transmembrane core. Both effects reduce the yield of functional receptor protein.