Concurrent silencing of TBCE and drug delivery to overcome platinum-based resistance in liver cancer

Platinum-based chemotherapy resistance is a key factor of poor prognosis and recurrence in hepatocellular carcinoma (HCC). Herein, RNAseq analysis revealed that elevated tubulin folding cofactor E (TBCE) expression is associated with platinum-based chemotherapy resistance. High expression of TBCE contributes to worse prognoses and earlier recurrence among liver cancer patients. Mechanistically, TBCE silencing significantly affects cytoskeleton rearrangement, which in turn increases cisplatin-induced cycle arrest and apoptosis. To develop these findings into potential therapeutic drugs, endosomal pH-responsive nanoparticles (NPs) were developed to simultaneously encapsulate TBCE siRNA and cisplatin (DDP) to reverse this phenomena. NPs (siTBCE + DDP) concurrently silenced TBCE expression, increased cell sensitivity to platinum treatment, and subsequently resulted in superior anti-tumor effects both in vitro and in vivo in orthotopic and patient-derived xenograft (PDX) models. Taken together, NP-mediated delivery and the co-treatment of siTBCE + DDP proved to be effective in reversing chemotherapy resistance of DDP in multiple tumor models.

Regulation of intracellular level of ATP and NADH in Escherichia coli to promote succinic acid production / 生物工程学报

Succinic acid is an important C4 platform chemical that is widely used in food, chemical, medicine sectors. The bottleneck of fermentative production of succinic acid by engineered Escherichia coli is the imbalance of intracellular cofactors, which often leads to accumulation of by-products, lower yield and low productivity. Stoichiometric analysis indicated that an efficient production of succinic acid by E. coli FMME-N-26 under micro-aeration conditions might be achieved when the TCA cycle provides enough ATP and NADH for the r-TCA pathway. In order to promote succinic acid production, a serial of metabolic engineering strategies include reducing ATP consumption, strengthening ATP synthesis, blocking NADH competitive pathway and constructing NADH complementary pathway were developed. As result, an engineered E. coli FW-17 capable of producing 139.52 g/L succinic acid and 1.40 g/L acetic acid in 5 L fermenter, which were 17.81% higher and 67.59% lower than that of the control strain, was developed. Further scale-up experiments were carried out in a 1 000 L fermenter, and the titer of succinic acid and acetic acid were 140.2 g/L and 1.38 g/L, respectively.

Biosynthesis of 2,5-dimethylpyrazine from L-threonine by whole-cell biocatalyst of recombinant Escherichia coli / 生物工程学报

2,5-dimethylpyrazine (2,5-DMP) is of important economic value in food industry and pharmaceutical industry, and is now commonly produced by chemical synthesis. In this study, a recombinant Escherichia coli high-efficiently converting L-threonine to 2,5-DMP was constructed by combination of metabolic engineering and cofactor engineering. To do this, the effect of different threonine dehydrogenase (TDH) on 2,5-DMP production was investigated, and the results indicate that overexpression of EcTDH in E. coli BL21(DE3) was beneficial to construct a 2,5-DMP producer with highest 2,5-DMP production. The recombinant strain E. coli pRSFDuet-tdh(Ec) produced (438.3±23.7) mg/L of 2,5-DMP. Furthermore, the expression mode of NADH oxidase (NoxE) from Lactococcus cremoris was optimized, and fusion expression of EcTDH and LcNoxE led to balance the intracellular NADH/NAD⁺ level and to maintain the high survival rate of cells, thus further increasing 2,5-DMP production. Finally, the accumulation of by-products was significantly decreased because of disruption of shunt metabolic pathway, thereby increasing 2,5-DMP production and the conversion ratio of L-threonine. Combination of these genetic modifications resulted in an engineered E. coli Δkbl ΔtynA ΔtdcB ΔilvA pRSFDuet-tdhEcnoxELc-PsstT (EcΔkΔAΔBΔA/TDH(Ec)NoxE(Lc)-PSstT) capable of producing (1 095.7±81.3) mg/L 2,5-DMP with conversion ratio of L-threonine of 76% and a yield of 2,5-DMP of 28.8% in 50 mL transformation system with 5 g/L L-threonine at 37 °C and 200 r/min for 24 h. Therefore, this study provides a recombinant E. coli with high-efficiently catalyzing L-threonine to biosynthesize 2,5-DMP, which can be potentially used in biosynthesis of 2,5-DMP in industry.

Delaying effect of pyrroloquinoline quinone on age-related skin aging in mice and its mechanisms / 中华皮肤科杂志

Objective:To investigate the effect of pyrroloquinoline quinone (PQQ) on age-related skin aging in mice and its mechanisms.Methods:Thirty Kunming mice were fed in specific pathogen-free condition, and equally divided into 3 groups: young group was fed with a normal diet for 8 months, old group was fed with a normal diet for 20 months to establish a mouse model of natural aging, and PQQ group was fed with PQQ-containing forages (4 milligrams of PQQ per kilogram of normal forages) for 20 months. After feeding, the mouse dorsal skin tissues were obtained, hematoxylin and eosin (HE) staining was performed to measure the epidermal and dermal thickness, Masson staining to detect changes in total skin collagen, immunohistochemical study to detect changes in expression of the proliferation marker Ki67, transmission electron microscopy to detect changes in autophagosomes in the mouse skin, and Western blot analysis to determine the expression of autophagy-related proteins Beclin1, LC3Ⅱ/LC3Ⅰ, and p62. Statistical analysis was carried out by using one-way analysis of variance for intergroup comparisons followed by least significant difference (LSD) - t test for multiple comparisons. Results:HE and Masson staining showed that the epidermal and dermal thickness and the percentage of area of dermis stained positive for collagen among the total area of dermis in the tested region were significantly lower in the old group (15.67 ± 0.36 μm, 87.95 ± 11.86 μm, 22.12% ± 1.72%, respectively) than in the young group (29.37 ± 0.25 μm, 264.93 ± 10.34 μm, 45.03% ± 1.54%, respectively, all P<0.05) , and significantly higher in the PQQ group (25.53 ±0.47 μm, 145.01 ± 9.71 μm, 31.17% ± 1.20%, respectively) than in the old group (all P<0.05) . Immunohistochemical study revealed that the expression of Ki67 was significantly lower in the old group (13.74% ± 3.06%) than in the young group (29.07% ± 2.79%, P<0.05) and PQQ group (21.20% ± 1.47%, P<0.05) . Transmission electron microscopy showed that the number of autophagosomes in the skin was significantly higher in the old group than in the young group ( P<0.05) , but significantly lower in the PQQ group than in the old group ( P<0.05) . As Western blot analysis revealed, the old group showed significantly decreased Beclin1 expression and LC3 Ⅱ/LC3 Ⅰ ratio, but significantly increased p62 expression compared with the young group (all P<0.05) ; compared with the old group, the PQQ group showed significantly increased Beclin1 expression and LC3Ⅱ/LC3Ⅰratio, but significantly decreased p62 expression (all P<0.05) . Conclusion:PQQ can delay the age-related skin aging in mice, likely by increasing the proliferative capacity of mouse skin cells and promoting skin autophagy.

Clinical features and tubulin folding cofactor E gene analysis in Iranian patients with Sanjad-Sakati syndrome / Características clínicas e análise genética e de cofatores de dobramento da tubulinaem pacientes iranianos com síndrome de Sanjad-Sakati

Abstract Objective Permanent hypoparathyroidism can be presented as part of genetic disorders such as Sanjad-Sakati syndrome (also known as hypoparathyroidism—intellectual disability-dysmorphism), which is a rare autosomal recessive disorder. Our aim was to confirm the diagnosis of a group of patients with dysmorphism, poor growth, and hypoparathyroidism clinically labeled as Sanjad-Sakati syndrome and to identify for the first time the genetic variations on Iranian patients with the same ethnic origin. Methods In this study, 29 cases from 23 unrelated Arab kindreds with permanent hypoparathyroidism and dysmorphism indicating Sanjad-Sakati syndrome were enrolled for 10 years in the southwest of Iran. The mutational analysis by direct sequencing of the tubulin folding cofactor E gene was performed for the patients and their families, as well as their fetuses using genomic DNA. Results Twenty-eight out of 29 cases had parental consanguinity. Twenty-seven cases presented with hypocalcemia seizure and two were referred because of poor weight gain and were found to have asymptomatic hypocalcemia. The dysmorphic features, hypocalcemia in the setting of low to normal parathyroid hormone levels and high phosphorus led to the diagnosis of these cases. Sequencing analysis of the tubulin folding cofactor E gene revealed a homozygous 12-bp deletion (c.155-166del) for all patients. Following that, prenatal diagnosis was performed for eight families, and two fetuses with a homozygous 12-bp deletion were identified. Conclusion These results make it much easier and faster to diagnose this syndrome from other similar dysmorphisms and also help to detect carriers, as well as prenatal diagnosis of Sanjad-Sakati syndrome in high-risk families in this population.

Resumo Objetivo O hipoparatireoidismo permanente pode estar presente como parte das doenças genéticas como na síndrome de Sanjad-Sakati (também chamada de síndrome de hipoparatireoidismo, retardo e dismorfismo), que é um distúrbio autossômico recessivo raro. Nosso objetivo foi confirmar o diagnóstico de um grupo de pacientes com dismorfismo, crescimento deficiente e hipoparatireoidismo clinicamente identificado como síndrome de Sanjad-Sakati e identificar as variações genéticas, pela primeira vez, em pacientes iranianos com a mesma origem étnica. Métodos Neste estudo, foram inscritos 29 casos de 23 famílias árabes sem parentesco com hipoparatireoidismo e dismorfismo indicando síndrome de Sanjad-Sakati, durante 10 anos no sudoeste do Irã. Foi feita a análise mutacional por sequenciamento direto do gene do cofator E de dobramento da tubulina dos pacientes e de suas famílias e também de seus fetos com o DNA genômico. Resultados Apresentaram consanguinidade parental 28 dos 29 casos. Desses, 27 casos apresentaram convulsão por hipocalcemia e dois foram encaminhados devido ao baixo ganho de peso, considerando diagnóstico de hipocalcemia assintomática. As características dismórficas, hipocalcemia na configuração de níveis de hormônio da paratireoide baixos a normais e alto nível de fósforo levaram ao diagnóstico dos casos. A análise de sequenciamento do gene do cofator E de dobramento da tubulina revelou deleção homozigótica de 12 pares de base (pb) (c.155-166del) em todos os pacientes. Após isso, foi feito o diagnóstico pré-natal em oito famílias e dois fetos foram identificados com deleção homozigótica de 12 pb. Conclusão Esses resultados tornam o diagnóstico dessa síndrome muito mais fácil e rápido do que outros dismorfismos semelhantes e também ajudam a detectar portadores, bem como o diagnóstico pré-natal da síndrome de Sanjad-Sakati em famílias de alto risco nessa população.

ATP regulation strategy and its application in the synthesis of microbial metabolites / 生物工程学报

Cofactor engineering, as a new branch of metabolic engineering, mainly involves ATP/ADP, NADH/NAD⁺, NADPH/NADP⁺ and other cofactors. Cofactor engineering can maximize metabolic flow by directly regulating the concentration and form of the cofactor of key enzymes in cells, and quickly direct carbon flow to target metabolites. ATP, as an important cofactor, is involved in many enzyme-catalyzed reactions in microbial cells, and leads to the restriction of the distribution of metabolic pathways by connecting or linking them into a complex network. Therefore, ATP regulation strategy is expected to be a favorable tool for industrial strain modification, to improve the concentration and production capacity of target metabolites, strengthen microbial tolerance to the environment and promote substrate utilization rate. The present review focuses on the recently used effective ATP regulation strategies and the effects of ATP regulation on cell metabolism in order to provide references for the efficient construction of microbial cell factories.

Differences in omega-5-gliadin allergy: East versus West

BACKGROUND: Omega-5-gliadin (O5G) allergy, also known as wheat-dependent exercise-induced anaphylaxis, is commonly reported in the Western, but not Asian, populations. Although significant differences in O5G allergy presentation across different populations are likely but there have been no previous reports on this important topic.OBJECTIVE: To report on the prevalence and characteristics of O5G allergy in Hong Kong (HK) compared with the United Kingdom (UK).METHODS: O5G allergy patients attending Queen Mary Hospital (HK cohort), and Guy's and St Thomas' Hospital, London (UK cohort) were studied and compared.RESULTS: A total of 46 O5G allergy patients (16 HK; 30 UK) were studied. In the HK cohort, 55% of all patients previously labeled as “idiopathic anaphylaxis” were diagnosed with O5G allergy. Exercise was the most common cofactor in both cohorts, followed by alcohol and nonsteroidal anti-inflammatory drugs (NSAID). A higher proportion of the HK cohort reported NSAID as a cofactor (13% vs. 0%, p = 0.048). In the HK cohort, more patients presented with urticaria and cardiovascular manifestations (100% vs. 77%, p = 0.036; 100% vs. 70%, p = 0.015, respectively); the range of presentation was more diverse in the UK cohort. In HK fewer patients adhered to wheat avoidance (50% vs. 87%, p = 0.007) and more patients avoided cofactors only (44% vs. 10%, p = 0.008).CONCLUSION: O5G allergy appears relatively underdiagnosed in HK. Urticaria and cardiovascular manifestations are common; NSAID plays an important role as a cofactor and patients are less concordant with dietary avoidance measures than in the Western population.

Cofactor engineering strategy for enhanced S-adenosylmethionine production in Saccharomyces cerevisiae / 生物工程学报

In order to study the role of cofactor engineering in enhancing the production of S-adenosylmethionine (SAM), we altered the form and concentration of cofactor in Saccharomyces cerevisiae through gene recombination. Effects of cofactor on product synthesis, carbon and energy metabolism were analyzed aiming to provide a theoretical basis for a successful metabolic engineering of SAM producing strains. Because NADPH metabolism in mitochondrion and cytoplasm of S. cerevisiae is relatively independent, the effect of intracellular NADPH availability on the production of SAM was studied in different compartments of S. cerevisiae BY4741. The expression of NADH kinase in mitochondria (POS5 encoded) and cytoplasm (POS5Δ17 encoded) was separately confirmed using a laser scanning confocal microscope. NADPH regulation strategy enhanced SAM production. Compared with the control strain, the intracellular SAM concentration of strain NBYSM-1 was increased by 3.28 times, and the intracellular SAM concentration of strain NBYSM-2 was increased by 1.79 times at 24 h fermentation. In addition, SAM titer and NADPH/NADP⁺ ratio in strain NBYSM-1 were significantly higher than that of strain NBYSM-2. Therefore, NADPH regulation strategy will be a valuable tool for SAM production and could further improve the synthesis of a large range of cofactor-driven chemicals.

New advances in phenylketonuria therapy research / 国际儿科学杂志

Phenylketonuria is a most common group of genetic metabolic diseases.Phenylketonuria is caused by enzymatic defects in the metabolic pathway,which is characterized by high blood phenylalanine concentration.Patients need early,reasonable treatment once diagnosis,otherwise there will be serious nervous system sequelae.Available treatments aim to decrease the blood phenylalanine concentration,reduce nervous system symptoms.The current primary treatment of phenylketonuria is the limitation of dietary phenylalanine intake.Considering the poor compliance with long-term eating restrictions and the heavy family burden,the application of new medicine such as trahydropterina cofactor,glycomacropeptide,large neutral amino acids can improve the therapeutic effect and living condition of phenylketonuria patients.In addition,recombinant phenylalanine ammonia lyase,hepatocyte transplantation,gene therapy,probiotics and other new treatments also seem to be a promising approach in the near future.

Busca por inibidores seletivos de Sirtuína 2 de T. cruzi empregando técnicas de planejamento de fármacos baseadona estrutura do receptor / Search for selective inhibitors of T. cruzi Sirtuin 2 employing drug design techniques based on receptor structure

A doença de Chagas, causada pelo parasita Trypanosoma cruzi, acomete entre 6 a 8 milhões de pessoas em todo o mundo. Conhecida como tripanossomíase americana, por ter sido considerada endêmica apenas na América Latina, esta doença, se espalhou para outros continentes devido aos movimentos migratórios se tornando um problema de sáude mundial. Estima-se que 56.000 novos casos e cerca de 12.000 mortes por complicações relacionadas à doença de Chagas anualmente. A quimioterapia disponível para o tratamento é composta apenas por dois fármacos, nifurtimox e benznidazol, no entanto são pouco eficazes na fase crônica da doença. Estes fármacos apresentarem, ainda, efeitos adversos graves e resistência por parte de algumas cepas do parasita. Diante deste panorama, é iminente a necessidade da busca de novos fármacos contra T. cruzi. Para a busca racional de novos quimiterapicos antiparasitários é fundamental a identificação e caracterização de vias metabólicas essenciais à sobrevivência dos parasitas. Assim, a enzima sirtuína 2 - Silent Information Regulator 2 (Sir2), tem importante papel para a infecção por T. cruzi, pois está totalmente envolvida no seu ciclo celular do parasita. Esta é uma enzima NAD+ dependente da classe III histona desacetilases, e se mostra como um interessante alvo bioquímico para o desenvolvimento de antichagásicos. A disponibilidade do sequenciamento genômico da Sir2 nos permite utilizar estratégias de planejamento de fármaco baseado no receptor (SBDD - Structure Based Drug Design) na identificação de candidatos a fármacos para essa doença. Entre as técnicas modernas de SBDD utilizadas, a triagem virtual possibilita identificar e selecionar inibidores enzimáticos potentes e seletivos para o alvo escolhido. Assim, neste trabalho, foi construído por meio da técnica de modelagem comparativa o modelo da enzima Sir2 de T. cruzi. Uma simulação por dinâmica molecular de 200ns, foi realizada para averiguar a estabilidade do modelo obtido. Diante da estabilização do modelo a partir de 100ns, o mesmo foi validado utilizando análise de clusters, RMSD (Root-mean-square Deviation) e análises de frequência de ligações de hidrogênio com o Cofator (NAD+) e os aminoácidos do sítio de catálise foram observadas, estes passos de simulação e validação foram realizados no programa DESMOND. Com o modelo robusto, os campos de interações moleculares (MIFs) foram gerados no programa GRID (Molecular Discovery v2.1) com o intuito de elucidar as regiões favoráveis a interação com a enzima em relação a propriedades físico-químicas da Sir2. A partir dos MIFs favoráveis a Sir2 de T. cruzi foi possível a construção de dois modelos farmacofóricos, o qual se baseou nas interações do Cofator (NAD+) e o sítio de catálise (Nicotinamida). O mesmo foi apliacdo como filtro para Triagem Virtual no programa UNITY da plataforma SYBYL X 2.0, utilizando os bancos de dados ZINC15 e GSK. A triagem resultou na seleção de 8 compostos candidatos a inibidores. Destes foram adquiridos 6 compostos por serem considerados mais promissores devido a complementariedade molecular. Estes foram testados contra a enzima de T. cruzi Sri2. Após o ensaio foi possível avaliar a potência de 4 compostos, sendo o composto CDMS-01 (IC50 = 39,9uM) o mais promissor que será submetido à processos de otimização molecular

Chagas disease, caused by the parasite Trypanosoma cruzi, affects between 6 and 8 million people worldwide. Also known as American trypanosomiasis, because it is considered endemic only in Latin America, but has spread to other continents due to migratory movements. It is estimated that 56,000 new cases and about 12,000 deaths from complications related to Chagas disease annually. The chemotherapy available for treatment consists of only two drugs, nifurtimox and benznidazole, however these are poorly effective in the chronic phase. These drugs also have serious adverse effects and resistance from strains of the parasite. Faced with this scenario, the need to search for new drugs against T. cruzi is imminent. For the drug planning for new antiparasitic chemotherapics, the identification and characterization of metabolic pathways essential to the survival of parasites is fundamental. Therewith, the sirtuin 2 - Silent Information Regulator 2 (Sir2) enzyme has an important role for T. cruzi infection, since Sir2 in the parasite is totally involved in its cell cycle. This is an NAD+-dependent enzyme of class III histone deacetylases, and it shows an interesting biochemical target for the development of antichagasic. The availability of Sir2 genomic sequencing allows us to use SBDD (Structure Based Drug Design) strategies in identifying drug candidates for this disease. Among the modern techniques of SBDD used, virtual screening makes it possible to identify and select potent and selective enzyme inhibitors for the chosen target. The model of the T. cruzi Sir2 enzyme was constructed using the comparative modeling technique. A molecular dynamics simulation of 200ns was performed to ascertain the stability of the obtained model. Considering the stabilization of the model from 100ns, it was validated using cluster analysis, Root-mean-square Deviation (RMSD) and hydrogen bond frequency analyzes with Cofator (NAD+) and the amino acids of the catalysis site were observed, these simulation and validation steps were performed in the DESMOND program. With the robust model, the molecular interaction fields (MIFs) were generated in the GRID program (Molecular Discovery v2.1) in order to elucidate the regions favorable to the interaction with the enzyme in relation to the physicalchemical properties of Sir2. From the MIFs favorable to Sir2 of T. cruzi it was possible to construct two pharmacophoric models, which was based on the interactions of Cofator (NAD+) and the catalysis site (Nicotinamide). It was also applied as a Virtual screening filter in the UNITY program of the SYBYL X 2.0 platform, using the ZINC15 and GSK databases. Screening resulted in the selection of 8 inhibitor candidate compounds. Six compounds were obtained from the screening, because they were considered more promising, and were tested against T. cruzi Sri2 enzyme. After the assay it was possible to evaluate the potency of 4 compounds, the most promising compound being CDMS-01 (IC50 = 39.9 µM) that will be submitted to molecular optimization processes

Manipulation of cofactor balance in microorganisms / 生物工程学报

Cofactor balance plays an important role in producing enzymes, pharmaceuticals and chemicals. To meet the demand of industrial production, microbes should maintain a maximal carbon flux towards target metabolites without fluctuations in cofactor. We reviewed the physiological function of cofactor and discussed detailed strategies to manipulate cofactor balance through biochemical engineering and metabolic engineering. Furthermore, we indicated future research needs to further regulate cofactor balance.

Validación de un ensayo automatizado para determinar la actividad de cofactor de ristocetina del factor von Willebrand / Validation of an automated assay to determine activity of ristocetin cofactor of von Willebrand factor / Validação de um teste automatizado para determinar a atividade de cofator de ristocetina do fator von Willebrand

La enfermedad de von Willebrand (EvW) se debe a un defecto, cuali o cuantitativo de la molécula del factor von Willebrand (VWF). Bajos niveles de VWF:Ag sugieren la EvW pero no distinguen los subtipos, por lo cual es necesario determinar también la funcionalidad del VWF para completar el diagnóstico. El método de referencia para estudiar la función del VWF es el ensayo del cofactor de ristocetina (VWF:RCo), basado en la habilidad del VWF para inducir la aglutinación de las plaquetas en presencia de ristocetina. Recientemente se han desarrollado métodos automatizados para determinar la actividad de cofactor de ristocetina. El objetivo fue evaluar el comportamiento del ensayo inmunoturbidimétrico automatizado VWF:RCo con el ensayo de actividad de VWF (VWF:Act) utilizado como screening de la EvW y con el ensayo tradicional de VWF:RCo por agregometría. La precisión intraensayo fue 3,1% y la precisión interensayo evaluada con el control normal fue de 3,2% mientras que con el control patológico se obtuvo un coeficiente de variación de 5,1%. Cuando se compararon los resultados de VWF:Act y VWF:RCo inmunoturbidimétrico en 60 pacientes, el coeficiente de correlación fue 0,96 con un bias -3,5%. En un subgrupo de 30 pacientes se comparó el VWF:Rco determinado por agregación y automatizada, y se obtuvo una correlación de 0,90 con un bias de 18,9%. Los valores de vWRCO obtenidos fueron, con un error total máximo permitido de 15%, estadísticamente comparables con aquellos determinados por el método VWF:Act y los valores de VWF:RCo obtenidos por agregometría en los pacientes estudiados.

Von Willebrand disease (VWD) is caused by a defective qualitative or quantitative von Willebrand factor (VWF) molecule. Low VWF:Ag suggests but does not distinguish VWD subtypes. Therefore, it is also necessary to determine the functionality of VWF to complete the diagnosis. The reference method to study VWF function is the ristocetin cofactor assay (VWF:RCo) based on the VWF ability to induce platelet aggregation in the presence of ristocetin. Recently, automated methods for determining the activity of ristocetin cofactor have been developed. The aim of this study is to evaluate the performance of VWF:RCo automated immunoturbidimetric assay: with VWF activity assay (VWF:Act ) used as a screening of VWD and the traditional VWF:RCo test by aggregometry. Intra-assay precision was 3.1% and interassay precision was: 3.2% in normal control and 5.1% low control: When VWF:Act and immunoturbidimetric VWF:RCo results were compared in 60 patients, the correlation coefficient was 0.96 with a -3.5% bias. In a subset of 30 patients VWF:Rco was compared, determined by aggregation and in an automated manner, yielding a correlation of 0.90 with an 18.9% bias. VWO:Rco values obtained were -with a 15% allowable total error- statistically comparable with those determined by the VWF:Act method, and the VWF:RCo values determined by aggregometry in the patients studied.

A doença de von Willebrand (DvW) ocorre devido a um defeito, qualitativo ou quantitativo da molécula do fator von Willebrand (VWF). Baixos níveis de VWF:Ag sugerem a EvW, mas não distinguem os subtipos, portanto é necessário determinar também a funcionalidade do VWF para completar o diagnóstico. O método de referência para estudar a função do VWF é o teste do cofator de ristocetina (VWF:RCo), baseado na habilidade de VWF para induzir a aglutinação das plaquetas em presença de ristocetina. Recentemente foram desenvolvidos métodos automatizados para determinar a atividade de cofator de ristocetina. O objetivo foi avaliar o comportamento do teste imunoturbidimétrico automatizado VWF:RCo com o teste de atividade de VWF (VWF:Act) utilizado como screening da EvW e com o ensaio tradicional de VWF:RCo por agregometria. A precisão intra-teste foi de 3,1% e a precisão inter-teste avaliada com o controle normal foi de 3,2% ao passo que com o controle patológico foi obtido um coeficiente de variação de 5,1%. Quando foram comparados os resultados de VWF:Act e VWF:RCo imunoturbidimétrico em 60 pacientes, o coeficiente de correlação foi de 0,96 com um Bias -3,5%. Num subgrupo de 30 pacientes se comparou o VWF:Rco determinado por agregação e automatizada, obtendo uma correlação de 0,90 com um bias de 18,9%. Os valores de VWF:Rco obtidos foram, com um erro total máximo permitido de 15%, estatisticamente comparáveis com aqueles determinados pelo método VWF:Act e os valores de VWF:RCo obtidos por agregometria nos pacientes estudados.

The influence of intracellular keratin 8 on hepatitis C virus replication / 药学学报

The level of intracellular keratin 8(KRT-8) is associated with liver diseases, whose expression is increased in hepatitis C virus (HCV)-infected patients with hepatocarcinoma and in cultural cells infected with HCV. However, it is not clear whether KRT-8 will impact HCV replication. In this paper, the HCV replication was analyzed in response to high expression and silence of KRT-8. The inhibitory activities against wild-type and mutant HCV were also analyzed by silence of KRT-8 or combined with known anti-HCV drug telaprevir. Results showed that the protein level of KRT-8 was increased in proportion with the HCV replication. The high expression was found to facilitate HCV replication, while the silence of KRT-8 was able to inhibit HCV replication and enhanced the anti-HCV activity of telaprevir. It also inhibited A156T and D168V mutant HCV, which are resistant to protease inhibitors. These results suggest that KRT-8 is a co-factor for HCV replication. Down-regulation of KRT-8 can inhibit wild type and mutant HCV replication to enhance the anti-HCV activity of known anti-HCV drugs. Therefore, KRT-8 may be a new target in the development of anti-HCV agents.

Complement and membrane-bound complement regulatory proteins as biomarkers and therapeutic targets for autoimmune inflammatory disorders, RA and SLE.

Complement system is a major effecter system of the innate immunity that bridges with adaptive immunity. The system consists of about 40 humoral and cell surface proteins that include zymogens, receptors and regulators. The zymogens get activated in a cascade fashion by antigen-antibody complex, antigen alone or by polymannans, respectively, by the classical, alternative and mannose binding lectin (MBL) pathways. The ongoing research on complement regulators and complement receptors suggest key role of these proteins in the initiation, regulation and effecter mechanisms of the innate and adaptive immunity. Although, the complement system provides the first line of defence against the invading pathogens, its aberrant uncontrolled activation causes extensive self tissue injury. A large number of humoral and cell surface complement regulatory protein keep the system well-regulated in healthy individuals. Complement profiling had brought important information on the pathophysiology of several infectious and chronic inflammatory disorders. In view of the diversity of the clinical disorders involving abnormal complement activity or regulation, which include both acute and chronic diseases that affect a wide range of organs, diverse yet specifically tailored therapeutic approaches may be needed to shift complement back into balance. This brief review discusses on the complement system, its functions and its importance as biomarkers and therapeutic targets for autoimmune diseases with focus on SLE and RA.

Correlation between overexpression of PC4 in lung adenocarcinoma with lymph node metastasis / 重庆医学

Objective To investigate the promotion effect of human transcriptional positive cofactor 4 (PC4) overexpression on lymphatic metastasis in lung adenocarcinoma .Methods 96 samples of lung adenocarcinoma tissue were collected .The immuno‐histochemistry(IHC) and real‐time quantitative polymerase chain reaction (qRT‐PCR) were adopted for detecting the expression levels of PC4 protein and mRNA .The correlation of PC4 expression with lymphatic metastasis and TNM stage was analyzed .Re‐sults The expression of PC4 protein was positively correlated mRNA in lung adenocarcinoma (r=0 .63 ,P<0 .01);the expression of PC4 protein was positively correlated with lymph node metastasis (χ2 =8 .29 ,P<0 .01) and TNM stage (χ2 =4 .71 ,P<0 .05);the expression of PC4 mRNA was also positively correlated with lymph node metastasis (χ2 = 8 .40 ,P< 0 .01) and TNM stage (χ2 =5 .10 ,P<0 .05) .Conclusion PC4 overexpression is found to be closely associated with the lymph node metastasis and TNM stage .PC4 may facilitate the lymph node metastasis of lung adenocarcinoma .

Validación de una técnica de actividad de factor von Willebrand que utiliza glicoproteína Ib mutada / Validation of a method based on a mutant glycoprotein Ib for von Willebrand factor activity measure / Validação de uma técnica da atividade do fator de von Willebrand que usa mutante de glicoproteína Ib

La medida de actividad del factor von Willebrand (vWF) es importante para el diagnóstico de la enfermedad de von Willebrand (vWD). El equipo Innovance®vWF Ac (Siemens Healthcare Diagnostics) utiliza un método inmunoturbidimétrico diseñado para coagulómetros Sysmex CA (Siemens). El objetivo del estudio fue desarrollar y validar la metodología del reactivo Innovance®vWF Ac para el coagulómetro ACL TOP 700 (Instrumentation Laboratory, IL) y realizar un estudio comparativo de su desempeño frente a la de la técnica vWF Activity (vWF Act, IL) y a la del cofactor de ristocetina (vWF:RCo) por agregación de transmisión de luz. Se desarrolló, calibró y validó la técnica mediante estudios de verificación de linealidad y precisión total para dos niveles de control. Se midió la actividad del vWF por los métodos Innovance®vWF Ac y vWF Act en 82 muestras consecutivas dentro del rango reportable. Para comparar métodos se utilizó la regresión de Deming. El análisis inicial de los resultados de Innovance®vWF Ac y vWF Act demostró que no eran estadísticamente comparables. Se observó un Bias negativo para Innovance®vWF Ac en muestras con vWF Act>150%. Excluyendo las mismas, se demostró que los resultados de ambas pruebas eran comparables, al igual que los de Innovance®vWF Ac y vWF:RCo en un subgrupo de 27 muestras. La adaptación del método Innovance®vWF Ac en el ACL TOP 700 fue exitosa, presentó linealidad y precisión acorde con los requerimientos de calidad del laboratorio, brindando resultados comparables a los de vWF Act y vWF:RCo en el rango de 5-150%, útil para el diagnóstico de vWD.

Measurement of vonWillebrand factor (vWF) activity is important for the diagnosis of von Willebrand disease (vWD). Innovance®vWF Ac assay was designed to Sysmex CA (Siemens) coagulometer. The aim of the study was to develop and validate a method for Innovance®vWF Ac in the ACL TOP 700 (Instrumentation Laboratory) coagulometer and to compare its performance against vWF Activity assay (Instrumentation Laboratory, vWF Act) and Ristocetin cofactor assay by light transmission aggregation (vWF:RCo). The method was designed, calibrated and validated through verification of linearity and total precision studies at two levels of control plasmas. vWF activity was measured by Innovance®vWF Ac and vWF Act in 82 consecutive samples with vWF Act values within the reportable range. For method comparison, Deming regression was used. The initial analysis of results showed that they were not statistically comparable with a negative Bias with Innovance®vWF Ac in samples with vWF Act>150%. Excluding these samples, Deming regression curve showed that both tests gave statistically comparable results, and also did Innovance®vWF Ac and vWF:RCo in a subset of 27 samples. The adaptation of the Innovance®vWF Ac method on the ACL TOP 700 coagulometer was successful, it met the quality requirements of the laboratory for linearity and accuracy, providing comparable results to vWF Act and vWF:RCo in the of 5-150% range, useful for vWD diagnosis.

Medir a atividade do fator von Willebrand (vWF) é importante para o diagnóstico da doença de von Willebrand (vWD). A equipe Innovance®vWF Ac (Siemens Healthcare Diagnostics) utiliza um método imunoturbidimétrico projetado para coagulômetros Sysmex CA (Siemens). O objetivo do estudo foi desenvolver e validar a metodologia do reagente Innovance®vWF Ac para o coagulômetro ACL TOP 700 (laboratório de instrumentação, IL) e realizar um estudo comparativo do seu desempenho perante a técnica vWF Activity (vWF Act, IL) e a do cofator de ristocetina (vWF:RCo) por agregação de transmissão de luz. A técnica foi projetada, calibrada e validada através de testes de verificação da linearidade e total precisão para dois níveis de controle. Foi mensurada a atividade do vWF pelos métodos Innovance®vWF Ac e vWF Act em 82 amostras consecutivas dentro do intervalo reportável. Para comparar métodos foi utilizada a regressão de Deming. A análise inicial dos resultados da Innovance®vWF Ac e vWF Act demonstrou que eles não eram estatisticamente comparáveis. Observou-se um viés negativo para Innovance®vWF Ac em amostras com vWF Act> 150%. Excluindo as mesmas, foi demonstrado que os resultados de ambos os testes eram comparáveis, do mesmo modo que os Innovance®vWF Ac e vWF:RCo num subgrupo de 27 amostras. A adaptação do método Innovance®vWF Ac no ACL TOP 700 foi bem-sucedida, apresentou linearidade e precisão conforme os requisitos de qualidade do laboratório, fornecendo resultados comparáveis aos vWF Act e vWF:RCo na faixa de 5-150%, útil para o diagnóstico da vWD.

Progress on molecular genetics in atypical hemolytic uremic syndrome / 国际儿科学杂志

Atypical hemolytic uremic syndrome(aHUS) has recently been shown to be a rare disease of genetic predisposition, including genes of complement factor H(CFH), membrane cofactor protein(MCP, CD46)and complement factor Ⅰ(CFI), which are complement regulatory genes. Genes mutation is about 50%,involving the three genes mutation, including nonsense mutation, missen mutation, silent mutation, splice mutation and insertion mutation. Autosomal dominant inheritance and autosomal recessive inheritance have been reported, however, for autosomal dominant inheritance, the three genes mutations are incomplete penetrance.

The Effects of Antidepressants on the Energy Metabolism in LETO Rat / 대한정신약물학회지

Objectives : A diverse range of adverse effects has been linked to the application of antidepressants for the treatment of depressive disorder. Recently, evidence has been emerging of the adverse metabolic effects of antidepressants. This study investigated the effects of antidepressants on plasma glucose and other factors in the fat and muscle tissue relating to metabolism. METHODS : Long-Evans-Tokushima-Ostuka (LETO) rats were used to evaluate the effects of different antidepressants. Amitriptyline, fluoxetine, and mirtazapine were administered to each of three subgroups for 4 weeks, between 11 and 15 weeks old, while a fourth subgroup was administered no antidepressant during the same period. Changes of weight and daily intake were monitored. Tissues and blood were collected at 15 weeks. RESULTS : The fluoxetine subgroup showed lower weight gain and lower food efficacy ratio than did the other subgroups. Blood glucose and other circulating factors showed no significant differences among groups, except for the leptin levels of the fluoxetine subgroup. However, the amitriptyline and mirtazapine subgroups showed similar patterns in the response of mRNA expression of peroxisome proliferator-activated receptors gamma cofactor-1 and uncoupling protein-1, 2, 3. CONCLUSION : These results could indicate possible differences in metabolic response based on the kind of antidepressant used.

Research Progress in Cofactor Engineering of Xylose Metabolism in Recombinant Saccharomyces cerevisiae / 中国生物工程杂志

Cofactor engineering, a vital part of metabolism engineering, changes the redox cofactor regeneration approach. Its main goal is to rebuild the components of metabolic products. The bioconversion of xylose for the production of ethanol is being studied intensively because ethanol is an alternative energy source and a potential liquid fuel. Saccharomyces cerevisiae has been traditionally used in producing ethanol from fermentable sugars but it cannot utilize xylose, only its isomer xylulose. Introduction of the xylose fermentation pathway from Pichia stipitis into S. cerevisiae enables xylose utilization in recombinant S. cerevisiae, but the ethanol yields of xylose fermentation with recombinant S. cerevisiae has been low and large amounts of the byproduct xylitol are produced. The major reason is that the catabolism of xylose with the fungal pathway leads an imbalance of redox cofactor. The process of the catabolism of xylose requires NADPH and NAD~+, both of which have to be regenerated in separated processes. More and more attention has therefore focused on the redox cofactor balance in S. cerevisia. The research progress of cofactor engineering to solve the imbalance of redox cofactor in xylose metabolism recombinant S. cerevisiae was introduced. This included expression of transhydrogenase, increasing the utilization of NADPH, and achieving the anaerobic reoxidation of NADH. Reversing the cofactor specificity of enzymes is another effective way.

Infección por micoplasmas en pacientes VIH-SIDA con enfermedad respiratoria / Infection caused by mycoplasmas in VIH-SIDA patients with respiratory disease

Se realizó un estudio prospectivo en 60 pacientes VIH/SIDA con manifestaciones respiratorias, en el período comprendido entre los meses de marzo a julio de 1998, encontrándose que 23 (38,3 %) fueron positivos para micoplasmas y la forma clínica que predominó en estos pacientes fue la infección respiratoria superior; de ellos fueron dados de alta mejorados 56,6 % y fallecieron 34,7 %, el conteo de linfocitos CD4+ menor que 200 células/mm³ se presentó en 69,6 % y el infiltrado bronconeumónico fue el hallazgo radiológico más frecuente. La coinfección con otras bacterias estuvo presente en 15 de los casos, predominando Pseudomona aeruginosa. La especie de micoplasma con mayor frecuencia encontrada fue Mycoplasma fermentans, y los pacientes infectados con esta especie estaban severamente inmunodeprimidos, además de colonizados por otros microorganismos.

A prospective study was carried out in 60 HIV/AIDS patients with respiratory manifestations, from March to July, 1998. 23 (38.3 %) proved to be positive for micoplasmas and the clinical form clinic prevailing in these patients was the upper respiratory infection. 56.6 % of them improved and were discharged, whereas 34.7 % died. The CD4+ lymphocites count under 200 cel/ mm3 was observed in 69.6 % and the bronchopneumonic infiltrate was the most frequent radiological finding. The co-infection with other bacterias was present in 15 of the cases, with a prevalence of Pseudomona aeruginosa. The species of mycoplasma with old opposing frequency was Mycoplasma fermentans was the mycoplasma species with the highest frequency found. The patients infected with this species were severely immunodepressed, besides being colonized by other microorganisms.