Recent progress of protein glycosylation characterization utilizing native conformer-resolved mass spectrometry / 中国药科大学学报

@#Glycosylation of proteins, one of the most prevalent and complex post-translational modifications occurring in nature, plays a crucial role in regulating protein net charge, conformation, binding properties and, ultimately, biological function.Traditional structural techniques are not amenable for glycoproteins due to the inherent heterogeneity of oligosaccharides.With the advances in analytical technique, mass spectrometry displays an increasingly crucial role in elucidating the structure of glycoproteins.Mass spectrometry-based proteomic technique can dissect the chemical composition and site information of low-abundance glycosylation at the peptide level.Instead, native mass spectrometry (nMS) can analyze intact glycoproteins while maintaining the information for glycan heterogeneity, and the insights into the regulatory effects of glycosylation on protein higher order structures and interactions with other proteins or ligands.As a representative structural mass spectrometry tool, ion mobility-based nMS strategy is powered by its conformer-resolving capability and by the feasibility of conformer manipulation through collision-induced unfolding.Consequently, native IM-MS analysis can provide rich information of dynamic protein conformations, allowing for the rapid identification and differentiation of protein isoforms in an unprecedented manner.In this review, we briefly introduced two emerging native IM-MS analytical modes, dynamic conformer-resolving mode and glycoform-resolving mode.Besides, we also discussed the recent progress of conformational and topological characterization of intact glycoproteins with three typical model systems based on two above-mentioned emerging modes of native IM-MS.

Chemical denaturants induced folding unfolding pathway of the recombinant zebrafish dihydrofolate reductase

Denaturation of proteins plays a crucial part in cellular activities. In this study, we have investigated the folding unfolding pathways of zebrafish dihydrofolate reductase (zDHFR) in presence of different chemical denaturants which were found to be an influential factor for the refolding yield by UV-visible spectrophotometric analysis. The activity change of zDHFR has been observed in presence of three different denaturants like Acetic Acid (AcOH), Sodium Dodecyl Sulphate (SDS), and Ethanol (C2H5OH). Spectrophotometric analysis reveals that protein unfolded completely at different concentrations and times by these denaturants. The spontaneous refolding experiments of chemically denatured zDHFR were also conducted to verify the spontaneous refolding yield. These investigations have helped us to decipher a picture about the denaturants contributing to achieving the refolding yield. We observed that acetic acid is a stronger denaturant among all, and the spontaneous refolding yields were higher from SDS denaturation. In the light of the above findings, higher spontaneous refolding yields were obtained from the low concentration of denaturants.

Anthropometric measurements of moderate concha-type microtia after auricular cartilage unfolding / 中国修复重建外科杂志

Objective: To explore the anthropometric changes of the auricle after auricular cartilage unfolding in moderate concha-type microtia patients, so as to provide the basis to help evaluate surgical timing and prognostic. Methods: A total of 33 children with moderate concha-type microtia, who were treated with auricular cartilage unfolding between October 2016 and September 2018 and met the inclusive criteria, were included in the study. There were 24 boys and 9 girls with an average age of 1.4 years (range, 1-3 years). Sixteen cases were left ears and 17 cases were right ears. The follow-up time was 12-23 months (mean, 17.5 months). The affected auricular detailed structures were observed and quantitatively analyzed before operation and at immediate after operation. The width, length, and perimeter of auricle before operation and at immediate after operation and at last follow-up were noted with three dimensional-scanning technology. The normal auricle was noted as control. Results: There were (7.5±1.0) and (11.3±0.8) structures of the affected auricle at pre- and post-operation, respectively, showing significant difference between pre- and post-operation ( t=23.279, P=0.000). The length, width, and perimeter of the affected auricle constantly increased after operation, and there were significant differences between pre-operation and immediately after operation and between immediately after operation and last follow-up ( P0.05). Conclusion: The auricular cartilage unfolding in treatment of the moderate concha-type microtia can receive more ear structures and increase auricle sizes, which make it possible for free composite tissue transplantation. In addition, the affected and the contralateral normal auricles have a very similar growth rate and it offers the theoretical foundation for the early treatment for moderate concha-type microtia.

Effects of Calcium on the Stability of VWF-A2 Domain by Molecular Dynamics Simulation / 医用生物力学

Objective To investigate the effect of calcium on the stability of VWF-A2 domain. Methods The crystal structures of A2 (not containing calcium) and A2/Ca2+ (with calcium bound) were downloaded from protein data bank. For A2 domain, the conformational changes, unfolding pathway differences and the exposure degree variance of cleavage sites caused by calcium binding were observed and analyzed by steered Molecular Dynamics simulations under constant force. Results The unfolding pathway of A2 domain and exposure process of cleavage sites were force-dependent. Calcium binding did not affect the unfolding process of A2 in the early stage. As the conformational rearrangement of α3β4-loop reduced its localized dynamic properties, the movement among β1-β4-β5 strands was restrained, which suppressed its further unfolding to stay in the intermediate steady state and delayed the cleavage-site exposure. Conclusions Stretch force could induce β5 strand of A2 unfolding and the cleavage-site exposure, while calcium binding inhibited ADAMTS13 proteolysis efficiency through stabilizing A2 hydrophobic core and covering its cleavage sites. These results way help to understand how ADAMTS13 cleavages the VWF-A2 domain and regulates the hemostatic potential of VWF, and further provide useful guidance on the design of related anti-thrombus drugs.

A Research on the Discovery Methods in Prescription and Medication Based on Professor Xue Boshou's Clinical Prescription Data / 世界科学技术-中医药现代化

How to discover the valuable knowledge from the large amount of prescription data accumulated in the long-term medical practice of traditional Chinese medicine (TCM) is one of the important contents of TCM modernization.Based on the prescription data of Xue' s clinical practice of many years,this paper explored the method of combining TCM network and prescription network to find out Xue' s common core drug combination.Based on 9,584 prescriptions in the Hospital Information System of Guang'anmen Hospital of China Academy of Chinese Medical Sciences,prescription network and drug network were constructed according to the similarity of prescription composition and drug co-occurrence relationship.Using the complex network analysis methods,such as community analysis method,to analyze the prescription and drug compatibility,the results were evaluated and analyzed by Xue and his successors.As a result,through complex network analysis,126 modular prescriptions and 4 TCM modules were obtained.One of the core components of the prescription module included Xiao Chai Hu decoction,Yin Qiao powder,and Sheng Jiang powder compound addition and subtraction.It was in consistent with the drug composition of exogenous febrile prescriptions excavated earlier.In conclusion,using the complex network methods,we can get some core drug combinations prescribed by Prof.Xue,and achieve the common compound core drug combination for treating diseases with certain vantages,laying a foundation for further inheriting and excavating Xue' s effective experience.

Psicoterapia psicanalítica focada nas emoções / Emotion-focused psychoanalytic psychotherapy

Este texto debruça-se sobre uma nova forma de fazer psicoterapia psicanalítica e psicanálise. A exploração das emoções como meio de mudança. Relato uma alteração na minha prática clínica em que a emoção passou a ser o foco da atenção. Uso um maior equilíbrio entre guiar e seguir o paciente, uma atenção aos marcadores e uso das intervenções para cada um desses marcadores como o desdobrar sistemático de uma evocação, focalização, ou o diálogo da cadeira vazia. São abordadas algumas dificuldades de integrar uma exploração mais eficaz da emoção num modelo psicanalítico praticado há muitos anos. Pequenas vinhetas clínicas ilustram a forma de explorar as emoções.

This text is about a new way of doing psychoanalytic psychotherapy and psychoanalysis. The exploration of emotions as a means of change. I report a change in my clinical practice in which emotion has become the focus of attention. I use a greater balance between guiding and following the patient, an attention to the markers and use of interventions for each of these markers as the systematic evocation unfolding, focusing, or the empty-chair dialogue. I describe some difficulties of integrating a more effective exploration of the emotion in a psychoanalytic model practiced for many years. Small clinical vignettes illustrate how to explore emotions.

Application of the Unfolding Model to the Aggression Dimension of the Dimensional Clinical Personality Inventory (IDCP) / Aplicación del Modelo de Desdoblamiento a la Dimensión de Agresividad del Inventario Dimensional Clínico de Personalidad (IDCP) / Aplicação do Modelo de Desdobramento à Dimensão Agressividade do Inventário Dimensional Clínico da Personalidade (IDCP)

This article compares the suitability of the dominance and unfolding models for the analysis of the Aggressiveness dimension in the IDCP (Dimensional Clinical Personality Inventory). The study included 975 subjects, with ages ranging from 18 to 81 years (M=29.82, SD=12.28), 58.9% of which were women. The IDCP is composed of 163 items and 12 dimensions; 27 items are related to Aggression. The analysis with the unfolding model indicated the exclusion of 15 items due to standard error. Results showed a better fit for the dominance model. This result may be due to the nature of the construct, because the items assess pathological aspects of personality representing one end of the continuum.

Este estudio compara la idoneidad de los modelos de dominancia y desdoblamiento para el análisis de la dimensión de Agresividad del IDCP (Inventario Dimensional Clínico de Personalidad). Participaron en el estudio 975 sujetos entre los 18 y los 81 años de edad (M=29,82; DE=12,28), de los cuales el 58,9% eran mujeres. El IDCP está integrado por 163 ítems y 12 dimensiones, con 27 ítems referentes a la Agresividad. El análisis a través del modelo del desdoblamiento produjo la exclusión de 15 ítems debido a error estándar. Los resultados mostraron un mejor ajuste del modelo de dominancia. Este resultado puede deberse a la naturaleza del constructo, por cuanto los ítems evalúan los aspectos patológicos de la personalidad que representan un extremo del continuum.

Este artigo compara a adequação da dominância e dos modelos que se desdobram para a análise da dimensão da agressividade no IDCP (Inventário Dimensional Clínico da Personalidade). O estudo incluiu 975 indivíduos, com idades que variam dos 18 até os 81 anos (M=29,82; SD=12,28), 58,9% dos quais eram mulheres. O IDCP é composto de 163 itens e 12 dimensões; 27 itens estão relacionados com a agressão. A análise com o modelo de desdobramento indicou a exclusão de 15 itens devido ao padrão de erro. Os resultados mostraram um melhor ajuste para o modelo de dominação. Esse resultado pode ser devido à natureza da construção, porque os itens avaliaram aspectos patológicos da personalidade, o que representa uma extremidade do continuum.

Effect of inactivation and reactivation conditions on activity recovery of enzyme catalysts

Enzymes are labile catalysts with reduced half-life time that can be however improved by immobilization and, furthermore, already inactivated catalyst can be recovered totally or partially, therefore allowing the large scale application of enzymes as process catalysts. In recent years a few studies about reactivation of enzyme catalysts have been published as a strategy to prolong the catalyst lifetime. Reported results are very good, making this strategy an interesting tool to be applied to industrial process. These studies have been focused in the evaluation of different variables that may have a positive impact both in the rate and level of activity recovery, being then critical variables for conducting the reactivation process at productive scale. The present work summarizes the studies done about reactivation strategies considering different variables: type of immobilization, enzyme-support interaction, level of catalyst inactivation prior to reactivation, temperature and presence of modulators.

Unfolding Ubiquitin by force: water mediated H-bond destabilization

Estudiamosel desdoblamiento forzado de una molécula de ubiquitina, usando elprotocolo de dinámica molecular “pull and wait” (PNW) a 300 K.Se implementó PNW en el programa CHARMM usando un tiempode integración de 1 fs y una constante dieléctrica de 1. La estructurasolvatada inicialmente, se colocó bajo tensión mecánica, ejerciéndosefuerzas en diferentes direcciones. El rompimiento de cinco enlacesde Hidrogeno entre los pliegues β1 y β5 que tiene lugar durante losprimeros 13 a 15 Å de extensión, marcan una barrera mecánica lacual define la máxima fuerza necesaria para el desdoblamiento. Lassimulaciones realizadas muestran que dado un tiempo adecuado, laaplicación de una fuerza pequeña puede desestabilizar los mencionadosenlaces de hidrógeno relativo a los enlaces que se pueden formar conmoléculas de agua; permitiendo la formación de enlaces de hidrógenoestables entre aguas y donadores o aceptores de la cadena principal.De esta forma, las simulaciones con PNW muestran que la tensiónmecánica no es responsable de separar los puentes de hidrógeno, estasólo los desestabiliza haciéndolos menos estables con respecto a losenlaces que se pueden formar con moléculas de agua. Simulacionesadicionales muestran que la fuerza necesaria para desestabilizarlos enlaces de hidrogeno, permitiendo su remplazo por enlaces conmoléculas de agua, depende fuertemente de la dirección de estiramiento.El protocolo de simulación que permite equilibrar a cada paso deextensión, nos evidenció eventos conducentes al desdoblamiento de lamolécula ubiquitina por fuerzas mecánicas...

Using the “pull and wait” (PNW) simulationprotocol at 300 K, we studied the unfolding of aubiquitin molecule by force. PNW was implemented inthe CHARMM program using an integration time step of1 fs and a uniform dielectric constant of 1. The ubiquitinmolecule, initially solvated, was put under mechanicalstress, exerting forces from different directions. Therupture of five hydrogen bonds between parallel strandsβ1 and β5 takes place during the extension from 13 to15 Å, defines a mechanical barrier for unfolding anddominates the point of maximum unfolding force. Thesimulations described here show that given adequatetime, a small applied force can destabilize those fiveH-bonds relative to the bonds that can be created towater molecules; allowing the formation of stableH-bonds between a single water molecule and the donorand acceptor groups of the interstrand H-bonds. Thus,simulations run with PNW show that the force is notresponsible for “ripping apart” the backbone H-bonds;it merely destabilizes them making them less stablethan the H-bonds they can make with water. Additionalsimulations show that the force necessary to destabilizethe H-bonds and allow them to be replaced by H-bondsto water molecules depends strongly on the pullingdirection. By using a simulation protocol that allowsequilibration at each extension we have been able toobserve the details of the events leading to the unfoldingof ubiquitin by mechanical force...

Estudamos o desdobramento forçado de uma molécula deubiquitina usando o protocolo de dinâmica molecular “pull and wait”(PNW) em 300 K. PNW foi implementado no programa CHARMMusando um tempo de integração de 1 fs e uma constante dielétrica de1. A estrutura solvatada, foi colocada sob estresse mecânico, exercendoseforças de diferentes direções. Simulações mostraram que a rupturade cinco ligações de Hidrogênio entre as dobras β1 e β5, que acontecedurante os primeiros 13 a 15 Å de extensão, define uma barreira, aqual determina a força máxima para o desdobramento. As simulaçõesmostram que, dado o tempo adequado, uma pequena força aplicadapode desestabilizar os mesmos cinco Hidrogênios relativos às ligaçõesH- que os grupos da cadeia principal podem fazer com a molécula deágua; permitindo a formação de ligações estáveis de H- entre moléculasde água e os grupos doadores e receptores da intercadeia. Desta forma,simulações que utilizam este protocolo mostram que a força nãoé utilizada para ®romper¼ as ligações H- da cadeia principal, apenasdesestabilizando-as e tornando-as menos estáveis do que as ligações queas mesmas podem fazer com a água. Simulações adicionais mostramque a força necessária para desestabilizar as ligações H- e permitir que asmesmas sejam substituídas por ligações do Hidrogênio com moléculasde água depende fortemente da direção da aplicação da força. Atravésda utilização de um protocolo de simulação que permite equilibrar emcada extensã, fomos capazes de observar os detalhes do mecanismo dedesdobramento da ubiquitina por força mecânica...

Detergent stable, halotolerant -amylase from bacillus aquimaris vitp4 exhibits reversible unfolding.

Halotolerant Bacillus aquimaris VITP4, isolated from Kumta coast (India), was used to produce extracellular α-amylase. The production was found to be maximal after 24 h of growth at pH 8.0 and 40 oC. Optimal activity of the purified enzyme was in the pH range of 7.5 – 9.5 at 40 oC. The enzyme was found to retain more than 60% of the initial activity even at NaCl concentration of 3.5 M, indicating that it is halotolerant. Calcium ion (0.01 mM) and CTAB (10 mM) enhanced the activity whereas EDTA decreased the enzymatic activity. Thermal inactivation kinetics suggested that the enzyme exhibits reversible unfolding even at high temperature (till 90 oC) and the t1/2 at 90 oC was found to be 43.5 min. These results suggests that the α-amylase from Bacillus aquimaris strain VITP4 is halotolerant, metal ion dependent enzyme which is stable in the presence of cationic detergent and moderate temperature conditions.

Unfolding for Color Volume Dataset Using the Difference of Segmented Contours / 대한의료정보학회지

OBJECTIVE: Unfolding is a rendering method to visualize organs at a glance by virtually incising them. Although conventional methods exploit gray-scale volume datasets such as CT or MR images, we use the Visible Korean Human dataset preserving actual color. This can be helpful for the study of anatomical knowledge. Segmented images of Visible Korean Human dataset store the boundary of organs. Since medical experts manually perform the segmentation from anatomical color images, it is very time-consuming. In general, therefore, some images selectively sampled with interval from entire color images are segmented. When we generate a segment volume dataset with the selected images, final results are deteriorated due to lack of segmentation information for missed images. In this paper, we solve this problem by generating intermediate images without performing a manual segmentation. METHODS: Firstly, after comparing differences of organ's contours in between two consecutive segmented images, we represent the differences as a user-defined value in the intermediate images. This procedure is repeated for all pairs of manually segmented images to reconstruct entire volume data consist of manually segmented images and their intermediate images. In rendering stage, we perform the radial volume ray casting along with the central path of target organ. If a ray reaches to a region having the user-defined values, we advance over the region without compositions to the boundary of that region. Then the color composition is begun by performing backtracking, since the advanced region is regarded to the thickness of it. RESULTS: As a result, we can produce high quality unfolding images for the stomach, colon, bronchus, and artery of the Visible Korea Human dataset. CONCLUSION: Since our approach can be applied to virtual dissection including actual human colors, it is helpful for the endoscopy and anatomy studies.

Two Conformations of Native Pseudomonas aeruginosa Apoazurin in Solution / 生物化学与生物物理进展

The mechanism of complex unfolding process of Pseudomonas aeruginosa apoazurin is an arguing problem. Recent published results indicated that this problem could be resolved by hypothesizing two native conformations coexisting in solution.Urea-induced unfolding of apoazurin was investigated further using intrinsinc fluorescence and CD spectra. Equilibrium unfolding curves in urea could be depicted with an apparent two-state transition, but a biphase kinetic process. The fast unfolding process is finished within a few seconds as monitored by stopped-flow fluorescence intensity, whereas the slow process requires several hours for as well as the circular dichroism spectra just after manual mixing of protein and denaturant could be simulated by superposition of the spectra of the native and fully unfolded protein with the same coefficient of 0.37. This strongly suggests the three-state mechanism with a partially unfolded intermediate on its pathway is inadequate. The hypothesis of two native conformations coexistence is a reasonable selection.

Unfolded Histidine-Tagged Protein is Immobilized to Nitrilotriacetic Acid-Nickel Beads, But Not the Nickel-Coated Glass Slide

The adsorption of proteins on the surface of glass slides is essential for construction of protein chips. Previously, we prepared a nickel-coated plate by the spin-coating method for immobilization of His-tagged proteins. In order to know whether the structural factor is responsible for the immobilization of His-tagged proteins to the nickel-coated glass slide, we executed a series of experiments. First we purified a His-tagged protein after expressing the vector in E. coli BL21 (DE3). Then we obtained the unfolding curve for the His-tagged protein by using guanidine hydrochloride. Fractions unfolded were monitored by internal fluorescence spectroscopy. The delta G(H20) for unfolding was 2.27 kcalmol +/- 0.52. Then we tested if unfolded His-tagged proteins can be adsorbed to the nickel-coated plate, comparing with Ni2+ -NTA (nitrilotriacetic acid) beads. Whereas unfolded His-tagged proteins were adsorbed to Ni2+ -NTA beads, they did not bind to the nickel-coated plate. In conclusion, a structural factor is likely to be an important factor for constructing the protein chips, when His-tagged proteins will immobilize to the nickel-coated slides.

The Thermodynamic Evidence of Two Native Conformations Coexisting in Solution For Apoazuirn: a DSC and CD Study / 生物化学与生物物理进展

Whether protein could adopt multiple conformations coexisting in solution is disputable. In a previous report,the conformation heterogeneity of apoazurin mutant M121L had been identified. The thermal unfolding of wild type apoazurin from Pseudomonas aeruginosa is re-investigated with differential scanning calorimetry (DSC) and circular dichroism (CD) methods. The results show that unfolding in the pH range from 4 to 9 is associated with two heat capacity maxima. The low temperature transitions are reversible at all pH conditions used, while the high temperature transitions are irreversible. The two unfolding transitions were analyzed by the two-interchangeable-conformation model with the fraction for the first transition (N1) from 64% at pH 4.0 to 55% at pH 9.0. Temperature induced unfolding monitored at 219 nm shows also two separate transitions. The ratio of the signal changes is consistent with the fractions obtained from the corresponding DSC measurements. These results provide further support for the hypothesis that at least two conformations of apoazurin coexists in solution.

Application of IRT Unfolding Model on "Autonomy in Learning Rating Scale for College Students" / 中国临床心理学杂志

Objective: Attempt to use generalized graded unfolding model to analyze the item of "Autonomy in Learning Rating Scale for College Students " adequacy of the scale designing and evidence probability of the usage of unfolding model in costructing tests. Methods: Based on randomly selected 600 graduates’ response of the scale, item analysis was carried on in 497 potent subjects. Results: Subjects whose trait continuum were between 2.5-4.0 and -3.5～-3.0 were short, and all the item locations were negative; all the item discriminations were above 0.5; the item information functions of 2, 6, 7, 19, 21, 25, 49, 50, 51 were below 0.4098; the fit plots of the item and test satisfied the requirements. Conclu- sion: Fit index of the whole scale is good; some items need to be revised or deleted; GGUM could be used in constructing attitude scales.

Isolation, characterization and effect of acidic pH on the unfoldingrefolding mechanism of serum albumin domains.

Three fragments, viz., BSA-CNBr1 – 183, BSA-CNBr184 – 582, and BSA-T377–582 representing domains I, II + III and III of bovine serum albumin have been isolated and purified. The physicochemical properties have been investigated and compared with their parent albumin molecule. The values of Stokes radii (nm) and intrinsic viscosities (ml/g) have been determined to be 2·36, 3·30; 3·43, 4·36; and 2·40, 3·13 for the fragments BSACNBr1– 183, BSA-CNBr184–582 and BSA-T377–582 respectively. The acid induced unfolding-refolding transitions of intact albumin and the fragment BSA-T 377–582 have been shown to occur in two steps while the fragments BSA-CNBr1 – 183 and BSACNBr184– 582 underwent single step transitions. The formation of the acid denatured states of intact albumin, BSA-CNBr1 – 183 and BSA-CNBr184–582 was accompanied by an increase of about 86, 56 and 44% in the values of intrinsic viscosities respectively. Since all the transitions were reversible, the values of equilibrium constants, KD, were calculated. The analysis of the dependence of KD on pH indicated that the first transition (N–X) of albumin was caused due to the uptake of about 3 protons by the native albumin. ,The intermediate state, X, is converted to acid unfolded state, D, by taking up another two protons. A comparision of the results on intact albumin with that of its fragments revealed that the second transition of the fragment BSA-T377– 582 and the two single step transitions of the fragment BSA-CNBr1–183 and BSA-CNBr184–582 were much closer to the second transition (X-D) of the intact albumin. The first transition of albumin has been attributed to its domain III represented by the fragment BSA-T377–582.