亚甲蓝光化学法病毒灭活血浆外泌体微小RNA差异表达谱分析 / 中国生物制品学杂志

@#Objective To explore the effect of methylene blue photochemistry(MB-P)viral inactivation treatment on the expression of microRNA(miRNA)in plasma exosomes,in order to provide a new reference for the quality control of MB-P virus inactivated plasma. Methods Whole blood samples of 11 healthy volunteers were collected from July 2021 to April2022. Fresh plasma from the same person was prepared into two parts,fresh frozen plasma(FFP)and MB-P virus inactivated plasma,respectively. The plasma exosomes were isolated by differential centrifugation,and identified by transmission electron microscopy(TEM)and nanoparticle tracking analysis(NTA). Then the expression profiles of miRNA were detected by microarray technique. Furthermore,four differentially expressed miRNA were verified by qRT-PCR,the target genes of differentially expressed miRNA were predicted by bioinformatics methods,and GO function enrichment analysis and KEGG pathway enrichment analysis were performed on the differentially expressed genes. Results The morphological characteristics and diameters of the extracted vesicles of the two groups were consistent with the characteristics of exosomes. Compared with the control group,there were 14 differentially expressed miRNA in plasma exosomes of MB-P group,of which the expression of six miRNA was up-regulated and eight miRNA was down-regulated. The results of qRT-PCR were generally consistent with the expression trend of microarray. The target genes of differentially expressed miRNA were mainly involved in DNA binding,ion binding,catalytic activity and other functions,and participated in a variety of biological processes such as nucleic acid metabolism,biosynthesis,and transcription regulation. In addition,significantly enriched functional pathways were closely related to viral infectious diseases,tumors,PI3K-Akt signaling pathway,MAPK signaling pathway and so on.Conclusion The expression of exosome miRNA in MB-P virus inactivated plasma was different from that in FFP. The plasma exosome miRNA may be used as a potential reference for the quality evaluation of MB-P virus inactivated plasma.

Metabolism and function of NK cells / 中国免疫学杂志

NK cells are important components of innate immune system and play a key role in immune responses.Activation of NK cells mainly depends on dynamic balance between activatory and inhibitory receptors expressed on surface.However,in many chronic diseases,balance between these receptors of NK cells is disorder,resulting in reduced cytolysis activity and cytokine produc-tion,which is in a state of immune inactivation.In recent years,many studies have shown that intracellular metabolism is crucial for immune cells such as NK cells,and changes of metabolism will regulate functions of immune cells by affecting cell development,pro-liferation and activity.In view of powerful anti-tumor and anti-viral effects of NK cells and their important clinical application value,it is important to study their metabolic characteristics and mechanisms.This review mainly introduces metabolic patterns of NK cell,related regulatory pathways,regulatory effects of metabolism on NK cell development,memory and functions,and metabolism-based NK cell therapy,also expounds important role of metabolism on NK cell biosynthesis,stability and effector function in vivo,as well as metabolism-related factors involved in NK cell inactivation in different chronic diseases,providing a solid research basis for clinical application of NK cell therapy.

Genetic analysis of a female child family with suspected X-linked recessive genetic disease / 中国热带医学

@#Objective　Genetic analysis was performed on a female child with chromosome Xq28 heterozygous deletion and suspected X-linked recessive disease to determine the morbidity and prognosis. Methods　A female child was admitted to the hospital on day 20 because of "jaundice for 20 days and difficulty in stopping bleeding at acupuncture sites". Low depth whole genome test of amniocentesis in late pregnancy suggested missing copy number of hemophilia A and X-linked mental retardation type 72. In order to further confirm the diagnosis and prognosis, peripheral blood of the children and their parents were collected for gene testing, chromosome inactivation test and genetic analysis. Results　Chromosome Xq28 of the child had 439.4 kb copy number heterozygous deletion variation, which was a clear disease-coding gene for functional loss included in ClinGen database. Chromosome inactivation test showed that the paternal X chromosome of the child was extremely inactivated. Haplotype analysis suggested that the normal chromosome of the subject was inherited from the mother, and there was heterozygous deletion on the paternal X chromosome, so it was inferred that the child will not develop disease or just have mild symptoms. Conclusion　It is necessary to analyze the X chromosome inactivation test for female patients with the pathogenic variation of X-linked recessive genetic disease to determine the possibility of the disease.

Optimization and application of caprylic acid precipitation in the purification of monoclonal antibody / 生物工程学报

In response to the market demand for therapeutic antibodies, the upstream cell culture scale and expression titer of antibodies have been significantly improved, while the production efficiency of downstream purification process is relatively fall behind, and the downstream processing capacity has become a bottleneck limiting antibody production throughput. Using monoclonal antibody mab-X as experimental material, we optimized the caprylic acid (CA) precipitation process conditions of cell culture fluid and low pH virus inactivation pool, and studied two applications of using CA treatment to remove aggregates and to inactivate virus. Based on the lab scale study, we carried out a 500 L scale-up study, where CA was added to the low pH virus inactivation pool for precipitation, and the product quality and yield before and after precipitation were detected and compared. We found that CA precipitation significantly reduced HCP residuals and aggregates both before and after protein A affinity chromatography. In the aggregate spike study, CA precipitation removed about 15% of the aggregates. A virus reduction study showed complete clearance of a model retrovirus during CA precipitation of protein A purified antibody. In the scale-up study, the depth filtration harvesting, affinity chromatography, low pH virus inactivation, CA precipitation and depth filtration, and cation exchange chromatography successively carried out. The mixing time and stirring speed in the CA precipitation process significantly affected the CA precipitation effect. After CA precipitation, the HCP residue in the low pH virus inactivation solution decreased 895 times. After precipitation, the product purity and HCP residual meet the quality criteria of monoclonal antibodies. CA precipitation can reduce the chromatography step in the conventional purification process. In conclusion, CA precipitation in the downstream process can simplify the conventional purification process, fully meet the purification quality criterion of mab-X, and improve production efficiency and reduce production costs. The results of this study may promote the application of CA precipitation in the purification of monoclonal antibodies, and provide a reference for solving the bottleneck of the current purification process.

Research progress on the pathogenesis of attention deficit hyperactivity disorder based on the theory of Dopamine deficits / 中华实用儿科临床杂志

Attention deficit hyperactivity disorder (ADHD) is a common neurodevelopmental disorder in childhood, which seriously affects physical and mental health in children.Its etiology and pathogenesis are complex and have not been fully elucidated.Currently, the theory of Dopamine (DA) deficits has been widely recognized and studied in the international academic community.The DA system is considered as the key to the pathogenesis of ADHD.The causes of DA deficits are complex.In addition to the well-established reuptake disorder caused by abnormal DA transporter function, DA deficits are also associated with the activation of DA vesicle cycle enzymatic inactivation, vesicle transport dysfunction, and receptor dysfunction, which are of great significance in analyzing disease pathogenesis and drug development.This article reviews the research on the causes of DA deficits proposed in recent years based on the theory of DA deficits, aiming to provide ideas and references for the research on the pathogenesis of ADHD in China.

Inactivación del virus Zika con medio de transporte de ácidos nucleicos, potencialmente aplicable a muestras clínicas para el diagnóstico molecular de SARS-CoV-2 / Inactivation of Zika virus with nucleic acids transport media, potentially applicable to clinical samples for the molecular diagnosis of SARS-CoV-2

RESUMEN Introducción: Los medios de colecta de muestras clínicas con capacidad de desnaturalizar virus reducen los riesgos de contagio durante el transporte y procesamiento. Objetivo: Emplear el medio de transporte de ácidos nucleicos (TAN) en muestras de exudado nasofaríngeo colectadas para el diagnóstico de SARS-CoV-2. Métodos: Se realizó un estudio experimental para demostrar la capacidad del medio de inactivar la infectividad viral. Se tomó como modelo de virus envuelto el virus Zika (VZk), cuyo nivel de bioseguridad es 2. Se evaluó el desempeño clínico del medio TAN para el diagnóstico de SARS-CoV-2. Se empleó una cepa del VZk propagada en la línea celular Vero y, previo a la infección de las células, el VZk se puso en contacto a intervalos de tiempo diferentes (2; 15 y 30 min) con el medio TAN puro; y luego se realizaron diluciones seriadas (10-1-10-4). La inactivación viral se evaluó por RT-PCR, en el sobrenadante y células colectadas, al culminar el periodo de propagación. El desempeño clínico del medio TAN se estimó tomando como referencia el CITOSWAB® VTM, en 30 exudados nasofaríngeos colectados para diagnóstico de la infección por SARS-CoV-2. Resultados: El VZk preservó su infectividad a diluciones del inóculo ≥ 10-2, independientemente del tiempo de contacto. La sensibilidad y especificidad clínica del medio TAN para el diagnóstico de SARS-CoV-2 fueron del 100 %, respectivamente. Conclusiones: Los resultados sugieren que muestras clínicas positivas a VZk en diluciones ≤ 10-1 del medio TAN pueden ser manipuladas de forma segura, lo que pudiera aplicarse potencialmente al diagnóstico molecular del SARS-CoV-2.

ABSTRACT Introduction: Collection media of clinical samples with the capacity to denature viruses reduce the risk of contagion during transportation and processing. Objective: To use the nucleic acids transport media (NATM) in nasopharyngeal swab samples collected for the diagnosis of SARS-CoV-2. Methods: An experimental study was conducted to demonstrate the medium capacity to inactivate viral infectivity. Zika virus (ZIKV), of biosafety level 2, was used as an enveloped virus model. The clinical performance of the NATM for the diagnosis of SARS-CoV-2 was evaluated. A ZIKV strain propagated in the Vero cell line was used and, prior to cells infection, ZIKV was in contact at different intervals (2; 15, and 30 min) with pure NATM; subsequently, serial dilutions (10-1-10-4) were performed. Viral inactivation was evaluated by RT-PCR in the supernatant and the collected cells when the propagation period was completed. CITOSWAB® VTM was used as reference to estimate the clinical performance of the NATM in 30 nasopharyngeal swabs collected for the diagnosis of SARS-CoV-2 infection. Results: ZIKV remained infectious at inoculum dilutions of ≥ 10-2, regardless of contact time. Clinical specificity and sensitivity of the NATM for the diagnosis of SARS-CoV-2 were 100%, respectively. Conclusions: Results suggest that ZIKV positive clinical samples at dilutions ≤ 10-1 of the NATM can be safely handled, which could potentially be applied to the molecular diagnosis of SARS-CoV-2.

Determination of methylene blue release in plasma virus inactivation bag by fluorescence energy transfer method / 中国输血杂志

【Objective】 To establish a simple, economical and rapid method for the determination of methylene blue (MB) release in virus inactivation bag. 【Methods】 Based on the fluorescence energy transfer between MB and BSA-stabilized gold nanoclusters (BSA-AuNCs), the standard curve of MB determination was established by measuring the fluorescence quenching degree of MB to BSA-AuNCs in different concentrations to conduct the determination of MB release in virus inactivation bag. 【Results】 There was a good linear relationship between the MB concentration (cMB) and the fluorescence quenching degree of BSA-AuNCs[ (I0-I)/I0=0.018cMB+ 0.021(r=0.996)] when the fluorescence emission wavelength was about 620 nm and the cMB was in the range of (0.9-36) μmoL/L. The recovery of MB was 98.00% -101.95 % when applied to determine MB at high, medium, and low concentrations, the obtained intra-day variation coefficients were 0.73%, 0.81% and 0.77% respectively, and the obtained inter-day variation coefficients were 3.92%, 3.81%, and 4.73% respectively. There was no significant difference between the results measured by this method and those measured by combination of solid-phase extraction and spectrophotometry(P>0.05). 【Conclusion】 The fluorescence energy transfer method could achieve simple and rapid determination of MB release in virus inactivation bag with accurate and reliable results.

Research progress on application of virus inactivation by ionizing radiation / 中国辐射卫生

Radiation sterilization is one of the most successful applications of ionizing radiation technologies. This paper reviews research on virus inactivation by ionizing radiation, focusing on its use in virus control for food, blood products, and homologous or heterologous tissue repair materials, inactivated viral vaccine preparation, and high-risk virus-related laboratory sample preparation, and also puts forward some thoughts on the application of ionizing radiation technologies in the prevention and control of coronavirus disease 2019.

A case of limbic encephalitis with positive anti-leucine-rich glioma inactivated 1 protein antibody and anti-myelin oligodendrocyte glycoprotein antibody / 中华神经科杂志

A case of limbic encephalitis with positive anti-leucine-rich glioma inactivated 1 protein (LGI1) antibody and anti-myelin oligodendrocyte glycoprotein (MOG) antibody was reported. The patient was a middle-aged male with a history of retinal vein occlusion. The main symptoms were temporal lobe epilepsy, facial arm dystonia, autonomic nerve dysfunction. Magnetic resonance imaging showed long T 2 signal in the right hippocampus without enhancement and normal perfusion. Electroencephalogram showed paroxysmal slow wave and sharp slow wave in interictal period. Blood anti-MOG antibody, blood and cerebrospinal fluid anti-LGI1 antibody were double positive. The main diagnosis was limbic encephalitis. After treatment with hormone and gamma globulin, the symptoms were improved and double antibodies were turned negative. Anti-LGI1/MOG double positive cases are rare, and the clinical manifestations and imaging manifestations of double positive antibody cases are not completely consistent with those with each single antibody, with different characteristics. This report can help clinicians enhance awareness.

Ozone disinfection for viruses with applications in healthcare environments: a scoping review

Abstract The aim of this scoping review was to provide sufficient information about the effectiveness of ozone gas in virus inactivation of surfaces and objects under different environmental conditions. The review was performed according to the list of PRISMA SrC recommendations and the JBI Manual for Evidence Synthesis for Scoping Reviews. The review was registered in Open Science Framework (OSF). EMBASE (Ovid), Lilacs, LIVIVO, MEDLINE (PubMed), SciELO, Scopus and Web of Science were primary sources, and "gray literature" was searched in OpenGray and OpenThesis. A study was included if it reported primary data on the effect of ozone gas application for vehicle-borne and airborne virus inactivation. No language or publication date restriction was applied. The search was conduct on July 1, 2020. A total of 16,120 studies were screened, and after exclusion of noneligible studies, fifteen studies fulfilled all selection criteria. Application of ozone gas varied in terms of concentration, ozone exposure period and the devices used to generate ozone gas. Twelve studies showed positive results for inactivation of different virus types, including bacteriophages, SARS-CoV-2 surrogates and other vehicle-borne viruses. Most of the studies were classified as unclear regarding sponsorship status. Although most of the population has not yet been vaccinated against COVID-19, disinfection of environments, surfaces, and objects is an essential prevention strategy to control the spread of this disease. The results of this Scoping Review demonstrate that ozone gas is promising for viral disinfection of surfaces.

Comparison of such three in vitro inactivation methods as alcohol inactivation, irradiation inactivation, and liquid nitrogen inactivation for the treatment of femur osteosarcoma in children / 中华实用儿科临床杂志

Objective:To retrospectively analyze the clinical effects and complications of alcohol inactivation, irradiation inactivation, and liquid nitrogen inactivation in the treatment of femur osteosarcoma in children, in an attempt to provide a theoretical basis for clinical selection of in vitro inactivation methods of tumor bone segment. Methods:The clinical data of 93 children with femur osteosarcoma admitted to the Department of Bone and Soft Tissue, the Affi-liate Cancer Hospital of Zhengzhou University from January 2008 to December 2017 were retrospectively analyzed, and 40 children, including 21 males and 19 females, aged 8-18 (13.65±2.87) years, who were treated with in vitro inactivation and replantation of autogenous tumor bone segment, were screened.Among these children, there was alcohol inactivation in 15 cases, irradiation inactivation in 12 cases, and liquid nitrogen inactivation in 13 cases.A comparison was drawn on these 3 inactivation methods with respect to bone healing time, bone healing rate, tumor recurrence rate, infection rate, fracture or fixation failure rate, and revision rate. Results:All those 40 children were subject to valid medical followed-up, with the alcohol inactivation group for (102.60±16.55) months, the irradiation inactivation group for (59.33±6.39) months, and the liquid nitrogen inactivation for (36.85±6.49) months.The difference in follow-up time of 3 groups was statistically significant ( P<0.05). Compared with other 2 groups, the index of bone healing time, bone healing rate, infection rate and revision rate in the alcohol inactivation group were unfavorable, which showed a significant difference (all P<0.05); However, there was no significant difference in the recurrence rate, fracture rate or fixation failure rate compared with other 2 groups (all P>0.05); There was no significant difference in all above indexes between the irradiation group and the liquid nitrogen group (all P>0.05). Conclusions:Three in vitro inactivation methods for the treatment of tumor bone segment are safe and reliable.The alcohol inactivated bone has a long healing time and more complications.Both irradiation inactivation and liquid nitrogen inactivation are clinical options, but irradiation inactivation requires professional equipment, which may limit the clinical application.

Case report of silent inactivation of Asparaginase in acute lymphoblastic leukemia with late bone marrow relapse / 中华实用儿科临床杂志

Retrospective analysis was performed on 1 child with silent inactivation (SI) of asparaginase (ASNas) who was diagnosed with acute lymphoblastic leukemia (ALL) and treated in the First Affiliated Hospital, Sun Yat-Sen University in October 2019.The patient was a 9 years and 3 months old boy who was diagnosed as ALL accompanied with late bone marrow relapse.After pegylated Escherichia coli-Asparaginase (PEG-ASNase) was given, he did not have the expected treatment-related adverse reactions, including hyperammonemia, hypofibrinogenemia, and the low activation of antithrombin Ⅲ (ATⅢ). The plasma asparagine (ASN) concentration failed to meet the depletion criteria and the ASNase activity was 64.5 U/L.Therefore, the SI of ASNase was confirmed.Erwinase was used to replace PEG-ASNase, the lowest level of ATⅢ was 33%, and the lowest level of fibrinogen was 1.20 g/L.Hyperammonemia and decreased ASN were also observed, and the ASNase activity was 1 813.0 U/L.All the above suggested that when, SI occurred, the replacement by Erwinase was effective.The ASNase activity should be monitored in ALL patients who were treated with ASNase.Monitoring the treatment-related adverse reactions such as hyperammonia and coagulation disorders closely has important implications to the SI of ASNase when the detection of ASNase activity was unavailable.

Effects of Virus Inactivation Treatment of Plasma Specimen on Plasma Concentration Determination of Vorico- nazole，Linezolid，Vancomycin and Teicoplanin / 中国药房

OBJECTIVE：To study the effects of virus in activation treatment of plasma specimen on plasma concentration determination of voriconzole ，linezolid，vancomycin and teicoplanin. METHODS ：The remaining plasma of 36 inpatients in our hospital after routine blood concentration examination of voriconazole ，linezolid，vancomycin and teicoplanin were collected as specimen（9 drug-contained plasma specimens for each drug ），and merged into three different concentration levels （low，medium， high）of mixed samples according the results of routine blood test. Then the mixed samples with different concentration levels were divided into inactivated group and non-inactivated group ，with 3 samples in each group. The inactivated plasma samples were heated at 56 ℃ for 30 min in metal bath with constant temperature. Non-inactivated group were not treated. After pretreating plasma sample of 2 groups，2-dimensional liquid chromatography was used to detect plasma concentration of the four drugs ；the difference of detection result between inactivated group and non-inactivated group were analyzed. RESULTS ：Plasma samples containing voriconazole，linezolid，vancomycin and teicoplanin were still stable after heating at 56 ℃ for 30 min in metal bath with constant temperature. Compared with non-inactivated group ，relative error of plasma concentration detection result of above 4 drugs were all lower than 15％ in low ，medium，high concentration mixed samples of inactivated group. CONCLUSIONS ：Plasma samples can be inactivated by heating at 56 ℃ for 30 min in metal bath with constant temperature ，when the plasma concentration of voriconazole，linezolid，vancomycin and teicoplanin are determined by 2-dimensional liquid chromatography.

Guidelines investigation and technical considerations of virus safety during investigational new drug application of innovative monoclonal antibody products / 药学学报

With the development of antibody manufacturing technology and improvement of new drug research in domestic industry, more innovative monoclonal antibody products submitted investigational new drug (IND) application. At the same time, monoclonal antibody products from abroad which have been approved marketing authorization and/or conducted clinical trials submitted IND applications in China. The National Medical Products Administration (NMPA) issued the "Guideline of Investigational New Drug Application" (No. 16, 2018) which emphasized the chemical, manufacturing, and control (CMC) regulatory, and dossier requirements in IND application, greatly promoted the application quality of innovative biological products. However, compared to the Food and Drug Administration (FDA) and European Medicines Agency (EMA), our particular guidelines are insufficient, such as guideline on virus safety evaluation of biotechnological investigational medicinal products. This review investigated the questions raised by sponsors from 2018 to 2020, including the end of production cell (EOPC) and/or unprocessed bulk (UPB) testing and virus removal or inactivation validation. Meanwhile, sponsors submitted different dossiers due to differences in understanding of stage requirements of guidelines from domestic and abroad. Based on the guidelines of virus safety from NMPA, FDA, and EMA, and the technical considerations, this review puts forward personal suggestions on the adventitious agents testing and virus removal or inactivation validation in manufacturing process, aim to ensure virus safety of innovative monoclonal antibody products in clinical trials.

Advances of pathogen inactivation technology in blood components and quality evaluation / 中国输血杂志

Through the screening of blood donors, detection of pathogen antigen and antibody, full coverage of nucleic acid detection, the risk of infectious blood transfusion has been reduced to a very low level. Especially, pathogen inactivation technology (PRT) has played an irreplaceable role in ensuring blood safety. The best way to inactivate pathogens is not only to effectively remove the target pathogens in the blood, but also to maintain the activity of active ingredients in the blood to the maximum extent, and it doesn′t affect the effect of blood therapy. In this paper, the development of pathogen inactivation technology is summarized, and the influence of pathogen inactivation treatment on the quality of blood components is discussed. It provides references for improving or developing new processing technology.

Effect of sample processing at 56℃ for 30 min on routine examination in Department of Blood Transfusion / 中国输血杂志

【Objective】 To investigate the effect of sample processing at 56℃ for 30 min on routine examination in Department of Blood Transfusion. 【Methods】 A total of 40 cross matched blood samples submitted by clinical departments of our hospital, were collected, and each sample was equally divided into two. Before and after heating at 56℃ for 30 min, the ABO blood group was detected by manual method and card method (gel card and glass beadle card), antibody titer was detected by coagulant method, and cross-matching was conducted by anti-globulin card method. Chi-square test and Wilcoxon signed rank test were used to compare the differences between the two groups (before and after heating treatment). 【Results】 The blood group detection rates of the experimental group were 100% (40/40), 37.5% (15/40) and 80% (32/40) by manual test tube method, gel card and glass beads card, respectively, P0.05). The matching rate of two groups of samples, cross-matched with corresponding donor samples, was both 100% (40/40) by coagulant method, and 100% (40/40) vs 25% (10/40) respectively by the antiglobulin card method (P<0.01). The other 30 samples in the experimental group presented weak agglutination in the secondary side. 【Conclusion】 The treatment of virus inactivation at 56℃ for 30 min has little effect on blood group identification by test tube method, antibody titer and cross-matching by coagulant method, and reduceds the occupational exposure of staff in Blood Transfusion Department.

Clinical Manifestation in Females with X-linked Metabolic Disorders: Genetic and Pathophysiological Considerations

Abstract Inborn errors of metabolism are predominantly autosomal-recessive disorders, but several follow an X-linked pattern of inheritance. They are called X-linked recessive, if the female carriers are asymptomatic, and are called X-linked dominant disorders, if almost all females are affected. Conditions, in which some females have symptoms while others are asymptomatic lifelong are simply referred to as X-linked. The aim of this review is to point out the variability in clinical manifestation of affected females in some X-linked metabolic disorders and to discuss on the basis of these examples possible mechanisms that may explain the broad phenotypic spectrum, such as the type of the underlying mutation, the issue of autonomous versus non-autonomous gene expression and the degree of skewing of X-inactivation. The use of the terms "X-linked dominant" and "X-linked recessive" will be discussed.

Effects of site-directed PEGylation on L-asparaginase thermostability / Efeitos da PEGuilação sítio-dirigida na termoestabilidade da L-asparaginase

The enzyme L-asparaginase (ASNase) is broadly applied as a drug to treat acute lymphoblastic leukemia, as well as in the food industry to avoid acrylamide formation in baked and fried food. In the present work, ASNase was covalently attached to polyethylene glycol (PEG) of different molecular weights (ASNase-PEG-5, ASNase-PEG-10, ASNase-PEG-20, and ASNase-PEG-40) at the N-terminal portion (monoPEGylation). Native and PEGylated forms were analyzed regarding thermodynamics and thermostability based on enzyme activity measurements. ASNase (native and PEGylated) presented maximum activity at 40 °C and denaturation followed a first-order kinetics. Based on these results, the activation energy for denaturation (E*d) was estimated and higher values were observed for PEGylated forms compared to the native ASNase, highlighting the ASNase-PEG10 with a 4.24-fold increase (48.85 kJ.mol-1) in comparison to the native form (11.52 kJ.mol-1). The enzymes were evaluated by residual activity over time (21 days) under different storage temperatures (4 and 37 °C) and the PEGylated conjugates remained stable after the 21 days. Thermodynamic parameters like enthalpy (ΔH‡), entropy (ΔS‡) and Gibbs free energy (ΔG‡) of ASNase (native and PEGylated) irreversible denaturation were also investigated. Higher - and positive - values of Gibbs free energy were found for the PEGylated conjugates (61.21 a 63.45 kJ.mol-1), indicating that the process of denaturation was not spontaneous. Enthalpy also was higher for PEGylated conjugates (18.84 a 46.08 kJ.mol-1), demonstrating the protective role of PEGylation. As for entropy, the negative values were more elevated for native ASNase (-0.149 J/mol.K), pointing out that the denaturation process enhanced the randomness and aggregation of the system, which was observed by circular dichroism. Thus, PEGylation proved its potential to increase ASNase thermostability

A enzima L-asparaginase (ASNase) é amplamente usada como medicamento para tratamento da leucemia linfoblástica aguda, bem como na indústria de alimentos para evitar a formação de acrilamida em alimentos cozidos e fritos. No presente trabalho, ASNase foi covalentemente ligada ao polímero poli(etilenoglicol) (PEG) de diferentes massas moleculares (ASNase-PEG-5, ASNase-PEG- 10, ASNase-PEG-20, and ASNase-PEG-40) na região N-terminal (monoPEGuilação) a fim de se estudar os efeitos da PEGuilação na termoestabilidade da enzima. As formas PEGuiladas e nativa foram analisadas em relação à termodinâmica e termoestabilidade a partir de atividade enzimática. A ASNase (nativa e PEGuilada) apresentou atividade máxima a 40 °C e a desnaturação ocorreu por cinética de primeira ordem. Com base nesses resultados, a energia de ativação para desnaturação (E*d) foi estimada e maiores valores foram observados para as formas PEGuiladas em comparação à enzima nativa, destacando-se a ASNase-PEG10 com aumento de 4.24 vezes (48.85 kJ.mol-1) em comparação com a forma nativa in (11.52 kJ.mol mol-1). As enzimas foram avaliadas por sua atividade residual ao longo do tempo em diferentes temperaturas de armazenamento (4 e 37 °C) e os conjugados PEGuilados mostraram-se mais estáveis após os 21 dias de ensaio. Parâmetros termodinâmicos como entalpia (ΔH‡) de desnaturação irreversível foram analisados. Valores maiores - e ), entropia (ΔS‡) de desnaturação irreversível foram analisados. Valores maiores - e ) e energia livre de Gibbs (ΔG‡) de desnaturação irreversível foram analisados. Valores maiores - e positivos - da energia livre de Gibbs foram encontrados para os conjugados PEGuilados (61.21 a 63.45 kJ.mol-1), indicando que o processo de desnaturação não ocorreu de forma espontânea. A entalpia também foi maior para os conjugados PEGuilados (18.84 a 46.08 kJ.mol-1), demonstrando o efeito protetivo da PEGuilação. Já para a entropia, os valores negativos foram mais elevados para a ASNase nativa (-0.149 J/mol.K), apontando que o processo de desnaturação aumentou a aleatoriedade e agregação do sistema, o que foi confirmado pelo dicroísmo circular. Dessa forma, a PEGuilação revelou o seu potencial de aumento de termoestabilidade para a ASNase

Asparaginase: an old drug with new questions

ABSTRACT The long-term outcome of acute lymphoblastic leukemia has improved dramatically due to the development of more effective treatment strategies. L-asparaginase (ASNase) is one of the main drugs used and causes death of leukemic cells by systematically depleting the non-essential amino acid asparagine. Three main types of ASNase have been used so far: native ASNase derived from Escherichia coli, an enzyme isolated from Erwinia chrysanthemi and a pegylated form of the native E. coli ASNase, the ASNase PEG. Hypersensitivity reactions are the main complication related to this drug. Although clinical allergies may be important, a major concern is that antibodies produced in response to ASNase may cause rapid inactivation of ASNase, leading to a worse prognosis. This reaction is commonly referred to as "silent hypersensitivity" or "silent inactivation". We are able to analyze hypersensitivity and inactivation processes by the measurement of the ASNase activity. The ability to individualize the ASNase therapy in patients, adjusting the dose or switching patients with silent inactivation to an alternate ASNase preparation may help improve outcomes in those patients. This review article aims to describe the pathophysiology of the inactivation process, how to diagnose it and finally how to manage it.

Hyphal Formation is Enhanced by 121°C Heat Inactivation of Serum Added to Culture Media

Background: A novel method for inducing hyphae formation, was described.Methods:Mycelia of experimental and clinical strains from 100 patients with oral lichen planuswere cultured in RPMI 1640 medium with 56°C or 121°C inactivated calf sera. After these strains were cultured for 2 h to 7 days, the average hyphae formation rate was observed under light microscopy. Results: The 121°C inactivated calf serum showed better outcomes than 56°C inactivated calf serum in terms of time and amounts of hyphae products. Conclusions:For Candida albicanshyphae culture, the 121°C inactivated serum is superior to the 56°C inactivated serum