Criofacoemulsificación en la cirugía de catarata / Cryophacoemulsification in cataract surgery

Objetivo: Demostrar los resultados de la criofacoemulsificación en la cirugía de catarata, como una mejora en la calidad visual y de vida de los pacientes. Métodos: Se realizó un estudio prospectivo y analítico de serie de casos en 43 ojos con catarata. Se utilizaron técnicas estadísticas descriptivas como media y desviación estándar. En los resultados no comparados la prueba de chi cuadrado, con significación del 95 por ciento y un valor de p < 0,05 se consideró estadísticamente significativa. Resultados: El 67,44 por ciento de los pacientes no sintieron dolor y lo refirieron muy leve en la colocación del blefaróstato, en la incisión por córnea clara y durante la colocación del lente intraocular; el 62,79 por ciento fueron féminas, mayores de 70 años; el 51,16 por ciento tenía dureza nuclear NO3 por LOCSIII; la visión de colores mejoró en el 90,70 por ciento; la sensibilidad al contraste mejoró en el 58,14 por ciento; el cilindro refractivo en el 72,09 por ciento fue menor de 0,5 dioptrías; la agudeza visual mejor corregida en el 93,02 por ciento fue de 0,8 a 1,0; la paquimetría demostró diferencias significativas al final; la pérdida celular endotelial media fue de 274,16 cel. /mm2; la hexagonalidad media se redujo en un 3,42 por ciento y la encuesta FV-14 fue muy buena en el 100 por ciento de los pacientes al mes de operado. Conclusiones: La criofacoemulsificación es segura y efectiva, que disminuye los efectos inflamatorios de la cirugía sobre la córnea, con alto grado de satisfacción de los pacientes(AU)

ABSTRACT Objective: Demonstrate the effect of cryophacoemulsification in cataract surgery as a way to improve patient visual quality and quality of life. Methods: An analytical prospective study was conducted of a case series of 43 eyes with cataract. Statistical analysis was based on mean and standard deviation descriptive techniques. In results not compared, a chi-square test with 95 percent significance and p < 0.05 was considered to be statistically significant. Results: Of the patients studied, 67.44 percent did not feel any pain and reported very mild pain during blepharostat placement, clear corneal incision and intraocular lens placement; 62.79 percent were females aged over 70 years; 51.16 percent had NO3 nuclear hardness by LOCSIII; color vision improved in 90.70 percent; contrast sensitivity improved in 58.14 percent; in 72.09 percent the refractive cylinder was smaller than 0.5 diopters; in 93.02 percent best corrected visual acuity was 0.8-1.0; pachymetry showed significant differences at the end; mean endothelial cell loss was 274.16 cel/mm2; mean hexagonality was reduced 3.42 percent, and the VF-14 index was very good in 100 percent of the patients one month after surgery. Conclusions: Cryophacoemulsification is safe and effective, reduces the inflammatory effects of surgery on the cornea and achieves a high level of patient satisfaction(AU)

Spirometry and Flow Volume Loops in Obstructive Sleep Apnea Patients

Introduction: Obstructive sleep apnea syndrome is the mostcommon sleep disordered breathing (SDB) and is associatedwith recurrent episodes of upper airway (UA) collapse duringsleep. Arousal from sleep is required to re-establish the airwaypatency. Patients with OSAS have various structural andfunctional abnormalities of the upper airway during sleep,which may reflect on their pulmonary function tests.Material and methods: We performed spirometry in 50diagnosed OSAS patients. Spirometric indices like FEF50/FIF50 >1 and saw tooth appearance of FV loops were studied.Their association with OSA and grades of OSA was evaluatedusing statistical analysis.Results: It was found that these spirometric indices werecommon in OSAS patients but their association with grades ofOSA were not statistically significant.Conclusion: Spirometry findings can point toward diagnosisof OSA but these findings are absent in most OSA patients.

Nuevas áreas de vida ­ Revisión muerte súbita 2018 / New areas of life ­ Sudden death review 2018

Una de cada cinco muertes en adultos en países desarrollados se debe a causas cardiovasculares; la mitad de esas muertes se produce de forma súbita y un gran porcentaje en el ámbito extrahospitalario. Las medidas de prevención se dividen en: aquellas destinadas a prevenir en primer lugar que el evento de muerte súbita cardíaca suceda, y aquellas cuyo objetivo es actuar en el momento en que el evento de muerte súbita está sucediendo. Las primeras tienen como objetivo disminuir las principales causas de muerte súbita en países desarrollados: las cardiopatías estructurales (cuya principal causa es la enfermedad coronaria). En este sentido, con el fin de intentar paliar el desarrollo de una cardiopatía que predisponga a la aparición de arritmias fatales y la MSC, se implementan medidas de prevención primarias higiénico-dietéticas y farmacológicas (con el objetivo de disminuir y el controlar los factores de riesgo) y, en aquellos con enfermedad cardiovascular ya establecida, se implementan las estrategias secundarias farmacológicas y/o quirúrgicas (revascularización, reemplazo valvular, etc.). El segundo abordaje surge del hecho de que, a pesar de todas estas medidas, un gran número de pacientes presentará eventos arrítmicos en el ámbito extrahospitalario (MSCEH), ya sea porque aunque recibieron el tratamiento óptimo presentan aún un elevado riesgo de MSC, porque no fueron diagnosticados a tiempo o porque a pesar de haber hecho estudios complementarios el diagnóstico es muy dificultoso. Existen dos estrategias: la primera son los dispositivos de cardiodesfibrilación implantables (o, más recientes, los chalecos vestibles). Estos aparatos están indicados para una población seleccionada, sea por haber presentado ya un episodio de muerte súbita abortado, o por presentar una cardiopatía (estructural o genética) que predisponga a una mayor probabilidad de sufrir un evento. La segunda estrategia es la educación y el desarrollo de programas de salud pública que permitan capacitar a la población general en la realización de RCP y el uso de desfibriladores automáticos externos (DEAs), los cuales deberían estar disponibles en cualquier lugar público. Múltiples estudios demostraron que el acceso de la población general al aprendizaje de maniobras de RCP sencillas y pragmáticas y la presencia de DEAs se traduce en un gran aumento de sobrevida sin secuelas en víctimas de MSCEH. (AU)

One of every five deaths in adults is due to cardiovascular causes, in developed countries, and half of these deaths will occur suddenly. A large percentage occur in the out of hospital setting, so measures to prevent it are divided into: those designed to prevent, in the first place, the sudden cardiac death event from happening and those whose purpose is to act when the sudden death event that has already occurred and it´s ongoing. The first aims to reduce the main causes of sudden death in developed countries: structural heart disease (with coronary heart disease as its main cause). In this regard, with the purpose to mitigate the development of a heart disease that predisposes the occurrence of fatal arrhythmias and SCD, we have primary prevention measures, like healthy life style conduct with or without pharmacological treatment, (whose objective is the reduction and control of cardiovascular risk factors) and, in those with cardiovascular disease already established, there is an implementation of pharmacological and / or surgical strategies (Revascularization, valve replacement, etc.). The second objective arises from the fact that, despite all these preventive and therapeutic measures, a large number of patients will present out-of-hospital cardiac arrest (OHCA) either because although they received optimal treatment they still remain in high risk of SCD, even because they were not diagnosed on time, or because despite having complementary studies made the diagnosis is very difficult. There are two well strategies: the first are implantable cardio-defibrillation devices (or, more recently, wearable vests). These are indicated for a selected population, either because they have already presented an episode of sudden aborted death, or because they have heart disease (structural or genetic), which predisposes to a greater probability of suffering an event. The second strategy is the education and development of public health programs that enable the general population to be trained in CPR and the use of external automatic defibrillators. (AEDs) should be available in any public place. Multiple studies showed that access to the general population for learning simple and pragmatic CPR maneuvers and the presence of AEDs is making an impact on a significant increase in survival without consequences in OHCA victims. (AU)

PHOTOSYSTEM II FLUORESCENCE AND GROWTH IN CABBAGE PLANTS (Brassica oleracea var. capitata) GROWN UNDER WATERLOGGING STRESS / FLUORESCENCIA DEL FOTOSISTEMA II Y CRECIMIENTO EN PLANTAS DE REPOLLO (Brassica oleracea var. capitata) EXPUESTAS A ESTRÉS POR ENCHARCAMIENTO

Waterlogging stress is a limiting factor in the production of crops grown in localities with high rainfall frequency. Waterlogging causes a decrease in the availability of O2 in roots, which substantially affects the anatomy, morphology and metabolism of plants. Oxygen deficiency inhibits mitochondrial respiration, oxidation, and oxygenation processes, causing severe affection of plant metabolism. A test in greenhouse conditions was carried out in Tunja, Colombia, in order to evaluate the effect of waterlogging on the growth of cabbage plants (Brassica oleracea var. capitata). Some plants were waterlogged for 25 days and their physiological response was compared with plants maintained at field capacity. As consequence of waterlogging, leaf area, total dry weight, chlorophyll content, leaf area ratio, absolute growth rate and relative growth rate were reduced. In addition, necrotic plants exhibited a high percentage of necrosis in the leaves. As for the variables related to chlorophyll fluorescence, there was a decrease of 17,9, 50,0 and 36,0% in the Fv/Fm values, ΦPSII and qP, respectively. All of these results indicate low tolerance of cabbage plants to waterlogging.

El estrés por encharcamiento es un factor limitante en la producción de algunos cultivos que se desarrollan en localidades, con alta frecuencia de lluvias. El encharcamiento causa disminución en la disponibilidad de O2 en las raíces, lo cual, afecta sustancialmente la anatomía, la morfología y el metabolismo de las plantas. El déficit de oxígeno inhibe la respiración mitocondrial, la oxidación y los procesos de oxigenación, con lo cual, el metabolismo de la planta resulta severamente afectado. Se desarrolló en Tunja, Colombia, un ensayo en condiciones de invernadero, con el propósito de evaluar el efecto del encharcamiento sobre el crecimiento de plantas de repollo (Brassica oleracea var. capitata). Algunas plantas fueron encharcadas durante 25 días y su respuesta fisiológica se comparó con la de plantas mantenidas a capacidad de campo. Como consecuencia del encharcamiento, se redujo el área foliar, el peso seco total, el contenido de clorofila y los valores de la relación de área foliar, de la tasa absoluta de crecimiento y de la tasa relativa de crecimiento. Adicionalmente, solo en las plantas expuestas al encharcamiento, se presentó necrosis en las hojas, en un alto porcentaje. En cuanto a las variables relacionadas con la fluorescencia de la clorofila, se registró una disminución de 17,9; 50,0 y 36,0%, en los valores de Fv/Fm, ΦPSII and qP, respectivamente. El conjunto de estos resultados indica baja tolerancia de las plantas de repollo al encharcamiento.

Expression, purification and biological effect of a novel single chain Fv antibody and protamine fusion protein for the targeted delivery of siRNAs to FGFR3 positive cancer cells

Background: Gain-of-function of fibroblast growth factor receptor 3 (FGFR3) is involved in the pathogenesis of many tumors. More and more studies have focused on the potential usage of therapeutic single-chain Fv (ScFv) antibodies against FGFR3. RNA interference (RNAi) has been considered as a promising therapeutic method against cancer. A tool which can deliver small interference RNAs (siRNAs) into FGFR3 positive cancer cells is very promising for anti-tumor therapy. Results: In this study, a novel fusion protein R3P, which consists of FGFR3-ScFv and protamine, was generated in Escherichia coli by inclusion body expression strategy and Ni-NTA chromatography. Its yield reached 10 mg per liter of bacterial culture and its purity was shown to be higher than 95%. 1 µg of R3P could efficiently bind to about 2.5 pmol siRNAs and deliver siRNAs into FGFR3 positive RT112 and K562 cells. Annexin V staining results showed that R3P can deliver the amplified breast cancer 1 (AIB1) siRNAs to induce RT112 cell apoptosis. Conclusion: These results indicated that R3P was a promising carrier tool to deliver siRNAs into FGFR3 positive cancer cells and to exert anti-tumor effect.

Parameters for the standardization of chlorophyll and assessment of PS II quantum efficiency for avocado (Persea americana Mill) / Parâmetros para a estandarização de índices de clorofila e da eficiência quantica do PS II em abacateiro (Persea americana Mill)

The standard physiological parameters for assessing stress physiology, of plants, such as chlorophyll index and photosystem II fluorescence, are essential for measuring reactions of plants to stress conditions. To help standardize the parameters for chlorophyll indices of chlorophylls a, b, and total and the fluorescence of photosystem II (Fv/Fm), which are physiological indicators of stress conditions, 6-mo-old seedlings of Persea americana Mill. cv. 'Duke7' and 'Toro canyon', were evaluated under shade house conditions. For each plant, chlorophyll indices were measured from the second through the fourteenth fully expanded leaves. Fluorescence was measured in the third, fifth, seventh, and ninth fully expanded leaf, and determined in function of both, time and intensity of the exposure light source, and the time for dark pre-acclimation of the leaf; was also compared right and left sides of the leaves. Chlorophyll indices were not different between the left and right sides of the leaves, but were different between varieties with 'Duke 7' having the highest value from the eighth leaf, while leaves tested for 'Toro canyon' did not show a difference. For Fv/Fm, there was an interaction among the three factors in both cultivars. A prolonged exposure time (nine seconds), short time for acclimation to darkness, and low intensity of exposure did not induce maximum fluorescence levels. On both avocado cultivars, exposure to maximum light intensity for 7 seconds after 25 to 30 minutes of acclimation to darkness, was the most favorable combination allowing the measurement of photosynthetic efficiency.

Os parâmetros padronizados para avaliação do estresse fisiológico de plantas, tais como índice de clorofila: fluorescência do fotossistema II, são essenciais para medir as reações de plantas a condições de estresse. Para auxiliar na padronização dos parâmetros para os índices de clorofila de clorofilas a, b e total e a fluorescência do fotossistema II (Fv / Fm), que são indicadores fisiológicos de condições de estresse, mudas de 6 meses de idade, de Persea americana Mill. Cv. 'Duke7' e 'Toro canyon' foram avaliadas sob condições casa de vegetação. Para cada planta, índices de clorofila foram medidos da segunda até a décima quarta folha completamente expandida. A fluorescência foi medida na terceira, quinta, sétima e nona folha totalmente expandida, e determinada em função do tempo e intensidade de exposição à fonte de luz, assim como do tempo para pré-adaptação da folha no escuro; também foram comparados entre o lado direito e esquerdo das folhas. Os índices de clorofila não foram diferentes entre os lados direito e esquerdo das folhas, mas foram entre diferentes variedades com 'Duke 7' tendo o valor mais alto a partir da oitava folha, enquanto que as de 'Toro canyon' não mostraram diferença. Para Fv/Fm, houve interação entre os três fatores em ambas as cultivares. O tempo prolongado de exposição (nove segundos), tempo curto para aclimatação no escuro, e baixa intensidade de exposição não induziu níveis máximos de fluorescência. Em ambas as cultivares de abacate, 100% de exposição à intensidade máxima de luz durante 7 segundos, após 25 a 30 minutos de adaptação no escuro, foi a combinação mais adequada para a medição da eficiência fotossintética.

In vitro aldose reductase inhibitory potential of fractions isolated from Potentilla fulgens roots.

The present study was investigated to identify the active fraction of P. fulgens with aldose reductase (AR) inhibitory potential. AR is the rate limiting step of polyol pathway implicated in the onset of chronic complications of diabetes. In this study, kidney homogenates of normoglycemic and diabetic mice were used as a source of AR enzyme preparation for in vitro analysis. The Terpenoid/Phenolic (TP) fraction of P. fulgens had the lowest IC50 value (0.152 mg/ml) for AR than the other fractions. TP fraction was separated using thin layer chromatography (TLC) and separated TLC fractions were tested for their AR inhibitory activity. Among the TLC fractions, F-V had the lowest IC50 value (0.156 mg/ml) and was characterized further using High Performance Liquid Chromatography (HPLC), Infra-Red (IR) Spectroscopy and Mass Spectroscopy (MS). F-V showed absorption maxima at λ230 nm and λ280 nm. HPLC profile of this fraction showed the presence of one prominent peak with a retention time of 1.621. IR spectra of the prominent peak indicated the presence of aromatic group which is phenolic in nature. MS of the prominent peak showed m/z ratio of 458.8. The active fraction isolated from P. fulgens has been shown to inhibit AR in normoglycemic and diabetic mice.

Expression and purification of soluble single-chain Fv against human fibroblast growth factor receptor 3 fused with Sumo tag in Escherichia coli

Background Overexpression or mutated activation of Fibroblast growth factor receptor 3 (FGFR3) is involved in the pathogenesis of many tumors. More and more studies focus on the potential usage of therapeutic antibodies against FGFR3. Results In this study, a novel single-chain Fv (ScFv) against FGFR3 was prepared and characterized. To achieve the soluble expression, ScFv was fused with Sumo (Small ubiquitin-related modifier) by polymerase chain reaction (PCR), and cloned into pET-20b. The recombinant bacteria were induced by 0.5 mM Isopropyl-ß-d-thiogalactopyranoside (IPTG) for 16 h at 20°C, and the supernatant liquid of Sumo-ScFv was harvested and purified by Ni-NTA chromatography. After being cleaved by the Sumo protease, the recombinant ScFv was released from the fusion protein, and further purified by Ni-NTA chromatography. The purity of ScFv was shown to be higher than 95% and their yield reached 4 mg per liter of bacterial culture. In vitro data showed that ScFv can significantly attenuate FGF9-induced phosphorylation of FGFR3. Conclusion We provide a novel method to produce soluble expression and bioactive functions of ScFv in Escherichia coli.

Teores de carboidratos e fluorescência da clorofila a em folhas de limeiras ácidas 'Tahiti' submetidas ao anelamento e incisão anelar de ramos / Levels of carbohydrates and chlorophyll fluorescence a leaves of acid lime trees 'Tahiti' subjected to girdling and ringing of branches

Objetivou-se estudar a dinâmica dos carboidratos e a fluorescência da clorofila a em folhas de limeiras ácidas 'Tahiti', submetidas a épocas diferentes de anelamento e incisão anelar de ramos. Foram estudadas quatro épocas de realização de anelamento e incisão anelar, com um tratamento adicional, sem interrupção do floema. O anelamento, independentemente da época de realização, aumentou os teores de amido (55 a 90%) e açúcares solúveis totais (8 a 18%) nas folhas do 'Tahiti' até por volta dos 50 aos 60 dias. A incisão anelar, independentemente da época de realização, aumentou a concentração de amido (10 a 35%) em relação à testemunha, porém, o aumento foi inferior ao que ocorreu nas plantas aneladas (55 a 90%). O anelamento promoveu aumentos de F0 e redução da relação Fv/Fm, sendo os valores máximos (350) e mínimos (0,78), respectivamente, atingidos por volta dos 60 dias. Para as plantas da testemunha e incindidas, houve aumento de F0, sendo o valor máximo atingido aos 50 dias (320), porém inferior aos observados para o anelamento, e não houve alteração da relação Fv/Fm .

The aimed to study the dynamics of carbohydrates and chlorophyll fluorescence a in leaves of acid lime trees 'Tahiti' subjected to different times girdling and ringing of branches. It was studied four times the realization of girdling and ringing, with an additional treatment, without phloem interruption. The girdling, regardless of the time of realization, increased starch (55 to 90%) and soluble sugars levels (8 to 18%) in 'Tahiti' leaves until around 50 to 60 days. The ringing, regardless of achievement time, increased starch content (10 to 35%) to the control compared, but increase was less than to that which occurred in girdled plants (55 to 90%). The girdling promoted F0 increase and Fv/Fm ratio reduced, being the maximum (350) and minimum (0.78), respectively, were reached at about 60 days. For control and ringing plants, there was F0 increase, being the maximum value was achieved in 50 days (320), but lower than those observed for girdling, and there was no change in Fv/Fm ratio.

Educational attainment and differences in fruit and vegetable consumption among middle-aged adults in the Korean National Health and Nutrition Examination Survey IV

We investigated whether socioeconomic differences affect fruit and vegetable (FV) consumption with respect to total intake and intake of various FV subgroups. Our study included 6667 adults aged 40-64 years who completed a dietary survey in the fourth Korean NHANES (2007-2009). FV intake was estimated from 24-hour recalls and food frequency questionnaires. Differences in FV consumption related to educational attainment were analyzed according to different nutritional categories of FV. Both men and women in the low-education group had the lowest intake of total FV and total fruits, and women also had the lowest intake of total vegetables. Also lowest in this group was consumption of mushrooms and vegetables (excluding kimchi) among men, and cruciferous and allium vegetables (excluding Chinese cabbage and radish) among women, while kimchi consumption was the highest in this group. Additionally, an association between educational level and intake of citrus fruits was evident among men. Adults in the low-education group consumed less carotene-rich FV, red fruit and/or vegetables, and dark-green leafy vegetables, fewer total vegetable dishes, and fewer types of fruit than in other groups. Men in this group had the lowest intake of yellow/orange fruit and/or vegetables, and women consumed the least folate-rich FV. There is a clear association between educational attainment and FV intake with regard to total intake, and to specific nutrients, bioactive compounds, colors, and variety.

Expression of a Functional zipFv Antibody Fragment and Its Fusions with Alkaline Phosphatase in the Cytoplasm of an Escherichia coli

BACKGROUND: Expression of recombinant antibodies and their derivatives fused with other functional molecules such as alkaline phosphatase in Escherichia coli is important in the development of molecular diagnostic reagents for biomedical research. METHODS: We investigated the possibility of applying a well-known Fos-Jun zipper to dimerize V(H) and V(L) fragments originated from the Fab clone (SP 112) that recognizes pyruvate dehydrogenase complex-E2 (PDC-E2), and demonstrated that the functional zipFv-112 and its alkaline phosphatase fusion molecules (zipFv-AP) can be produced in the cytoplasm of Origami(DE3) trxB gor mutant E. coli strain. RESULTS: The zipFv-AP fusion molecules exhibited higher antigen-binding signals than the zipFv up to a 10-fold under the same experimental conditions. However, conformation of the zipFv-AP seemed to be influenced by the location of an AP domain at the C-terminus of V(H) or V(L) domain [zipFv-112(H-AP) or zipFv-112(L-AP)], and inclusion of an AraC DNA binding domain at the C-terminus of V(H) of the zipFv-112(L-AP), termed zipFv-112(H-AD/L-AP), was also beneficial. Cytoplasmic co-expression of disulfide-binding isomerase C (DsbC) helped proper folding of the zipFv-112(H-AD/L-AP) but not significantly. CONCLUSION: We believe that our zipFv constructs may serve as an excellent antibody format bi-functional antibody fragments that can be produced stably in the cytoplasm of E. coli.

Screening, expression and characterization of single chain Fv (scFv) against basic fibroblast growth factor / 中华微生物学和免疫学杂志

Objective To obtain the specific human scFv basic fibroblast growth factor(bFGF)using phage antibody library technology. Methods The library was panned with human recombinant bFGF for 4 rounds. The antigen binding activities of random clones were tested by ELISA in order to select specific antibodies, which were then examined by DNA sequence analysis. Results The positive clone selected from the 104 random clones was able to bind bFGF specifically, while not able to bind other growth factors,such as aFGF, VEGF(vascular endothelial growth factor). By competition ELISA assay we found one clone 44 could inhibit bFGF binding to FGFR1. Conclusion Seven specific human phage antibody against bFGF was obtained by phage display technique, one clone could inhibit bFGF binding to its high affinity receptor FGFR1.

Rational design, expression and biological activity assessment of a novel peptide based on ricin toxin antibody / 中华微生物学和免疫学杂志

Objective To design and express a novel peptide based on ricin toxin antibody in E. coli, and to evaluate its biological activity. Methods Based on the crystal structure of ricin toxin A chain (RTA) and the RTA-rRNA interact in the complex model, the steric conformation of RTA was theoretical modeled and its functional domain was preliminarily determined. The humanized single-domain RTA antibody was designed rationally by computer-guidod molecular design method. Its coding sequence was ob- tained by overlapping extension PCR, and cloned into the pET-32a vector. The fusion protein was then ex-pressed in E. coli BL21 (DE3), identified by Western blot, and purified with Ni-NTA agarose. The binding and neutralizing activity of this novel peptide for riein was evaluated by competitive ELlSA assay and MTT assay. Results A recombinant human single-domain antibody expressing a polypeptide against RTA in the CDR3 loop was designed. The fusion protein was successfully expressed in E. coll. The purified protein can bind to ricin, and neutralize its activity in SP2/0 viability assay. Conclusion The success of the novel pep-tide based on riein toxin antibody provides a novel method to develop new generation of ricin antagonists.

Construction and Verification of High-capacity Ribosome Display Single-chain Fv Library / 中国实用内科杂志

Objective To construct high-capacity ribosome display single-chain Fv library for selection of high affinity ScFv antibody.Methods We isolate human lymphocyte from peripheralblood(2 normal,3 gastric cancer,3 colonic cancer,1 pancreatic cancer,each 5 mL and 2 newborn,each 2 mL)and extract RNA for cloning whole human heavy chain and light chain gene by RT-PCR.VH and VL were rearranged randomly by SOEing(splicing by overlap extension,SOEing).Finally,the elements for in vitro screening such as T7 promoter and ribosome binding site were introduced while the SOEing products were amplified.Moreover,ribosome display template were verified by blue/white screening and further sequencing.Results We successfully constructed ribosome display ScFv library with a volume of 1.1?1013.Conclusion The construction of high-capacity ScFv library shed light on multiple therapeutic ScFv screening.

Preparation and Identification of Anti-lactoferrin Single Chain Fv Antibody / 医学研究杂志

Objective To prepare a specific anti-lactoferrin single chain variable fragment(ScFv).Methods Anti-lactoferrin clones were screened from a 'naive' phage antibody library against the immobilized lactoferrin antigen,then the clones were transformed to the E.coli HB2151 to give soluble expression of antibody fragments.The culture supernatant containing ScFv was purified by immobilized metal affinity chromatography,and then determined with SDS-PAGE and ELISA.Results The results demonstrated that ScFvs were specific;they did not react with transferrin,lysozyme and bovine serum albumin in ELISA.The SDS-PAGE showed that the ScFvs had high purity through affinity chromatography and the molecular weight of them was about 32 kD.Conclusions The successful generation of the ScFvs against lactoferrin provides a basis for further study and clinical applications for dry eye and other ocular diseases.

Nanocomoposite probes composed of fluorescent magnetic nanoparticles and PSA ScFv antibody for targeted imaging and therapy of prostate cancer / 中国肿瘤生物治疗杂志

Objective:To investigate the feasibility of targeted imaging and therapy of prostate cancer using nanocomposite probes composed of fluorescent magnetic nanoparticles(FMCNPs) and single chain Fv(ScFv) antibody specific for gama-seminoprotein.Methods:The nanocomposite probes(FMCNPs-ScFv) were prepared by conjugating fluorescent magnetic nanoparticles with singlegama-chain Fv antibody specific gama-seminoprotein,and were characterized by high resolution transmission electron microscopy,fluorescent spectrum and magnetic spectrum.Nanocomposite probes were incubated with prostate cancer LNCaP cells,and the targeting results of nanocomposite probes were observed by fluorescent microscopy.The cytotoxicity effect of the nanocomposite probes was measured by MTT.Nude mice models of prostate cancer were established and identified by immunohistochemistry method.The nanocomposite probes were injected into nude mice via tail vein.The distribution of nanocomposite probes in the nude mice was observed by Micro-animal imaging system,targeted imaging of the prostate cancer was observed by MR instrument.The nude mice with prostate cancer were irradiated with 100 W magnetic field for 30 min,and the changes of tumor sizes were observed.Results:The FMCNPs-ScFv nanocomposite probes were successfully prepared.Nanocomposite probes entered into the cytoplasm of cancer cells and exhibited low cytotoxicity effect.Nude mice model with prostate cancer were successfully fabricated;the nanocomposite probes distributed quickly in the main organs of mice,and gradually concentrated on the tumor tissues within 24 h.MR images showed that the tumor images were gradually enhanced from 6 h to 24 h after injection of the nanocomposite probe.Four days after magnetic irradiation,the tumors in the nude mice grew slower compared with the control nude mice(P

THE CONSTRUCTION AND EXPRESSION OF THE MURINE SCFV GENE IN E. COLI AGAINST HUMAN CERVICAL CANCER / 药物分析学报

Objective To obtain the gene of murine Single chain Fv fragment (ScFv) against human cervical cancer and to express it in E. coli. Methods The variable region gene fragments of the heavy and light chains, which were amplified respectively using recombinant DNA techniques from CsA125 hybridoma cells, were spliced together through a flexible linker to ScFv against human cervical cancer. The ScFv genes were then cloned into expression vector pCANTAB 5E and expressed in E. coli HB2151 and TG1 respectively. The soluble ScFv were characterized by SDS PAGE and Western blot. The antigen-binding activities of the soluble and phage displayed ScFv were assayed by ELISA and cell immunohistochemical analysis. Results The expressed ScFv antibodies were soluble and phage displayed. The soluble ScFv secreted and expressed in E. coli HB2151 induced by IPTG were confirmed with SDS-PAGE, Western blot and ELISA. The specific binding capacity of the soluble and phage displayed ScFv to the surface associated antigen of human cervical cancer cell line was further confirmed with immunohistochemical studies. Conclusion The soluble and phage displayed ScFv expressed in E. coli against human cervical cancer showed high, specific affinity for the cervical cancer cell line surface associated antigen.

Factor V Leiden mutation in Korean women with pregnancy-induced hypertension / 대한산부인과학회지

OBJECTIVE: The purposes of this study was to evaluate the frequency of Leiden mutation (missense mutation in the factor V gene at exon 10, 1691 CGA to CAA) in Korean women with well characterized pregnancy-induced hypertension (PIH) compared with normotensive gravid women. METHODS: Genomic DNA from 121 PIH cases and 98 normotensive pregnant control cases were used for polymerase chain reaction (PCR). To genotype Leiden mutation (missense mutation in the factor V gene, exon 10 (1691 G to A)), primers (5'-TGC CCA GTG CTT AAC AAG ACC A-3', 5'-TGT TAT CAC ACT GGT GCT AA-3') were employed to make 267 base pair (bp) PCR product. There was an initial denaturation at 94 degrees C 5 min, followed by 30 cycles of one minute at 94 degrees C, one minute at 55 degrees C, and one minute at 72 degrees C. A 267 bp PCR product was further digested with Mnl I for 2 hour at 37 degrees C and analysed through 12% polyacrylamide gel electrophoresis to determine genotype. Allele 1691G yielded 37 bp, 67 bp, 163 bp fragment and allele 1691A yielded 67 bp, 200 bp fragment. RESULTS: We examined the genotypes of factor V of 121 Korean women with pregnant induced hypertension and 98 normal pregnant women. None of the 219 Korean women carried the factor V Leiden mutation. CONCLUSION: The factor V Leiden mutation is absent and not a common cause of PIH in Korean women.

Preparation and characterization of disulfide stabilized Fv fragment antibody against N-terminal fragment of human lipopolysaccharide binding protein / 第三军医大学学报

Objective To obtain the disulfide stabilized Fv fragment (dsFv) against N-terminal fragment of human lip polysaccharide binding protein (NH-LBP) and to identify its biological vitality. Methods The disulfide stabilized Fv fragment antibody (dsFv) was obtained after the inclusion bodies of dsFvVH and dsFvVL had been refolded and purified. Then the characteristics of dsFv were determined in vitro by ELISA and by detecting the secretion of tumor necrosis factor alpha (TNF-?) in rats. Results There was 2.1 mg protein of dsFv obtained. dsFv had good combination with NH-LBP and could restrain inflammatory reaction caused by lip polysaccharide (LPS) in vivo. Conclusion It is feasible to get dsFv against NH-LBP by respective expression of VL and VH. The partial inhibition of the biological function of LBP by dsFv is a new way to restrain the over-inflammatory reaction in vivo.

Reconstruction,expression and characterization of dsFv V_L of human antibody to N terminal fragment of human lipopolysaccharide binding protein / 第三军医大学学报

Objective To introduce the mutated gene coding cysteine into the gene of dsFv VL of human antibody to N terminal fragment of lipopolysaccharide binding protein(LBP)and to express,purify the mutated dsFv VL in bacterium.Methods We reconstructed and sequenced the mutated gene of VL of human mAb Fab to LBP by Mega-primer PCR based on point mutagenesis method.Some codes of FWR1 of VL had been replaced by TGT in order to code cysteine.The DNA sequence of reconstructed VL was inserted into vector pET-28a(+),then VL was expressed by E.coli.BL21 star(DE3)and was purified by chromatography.Finally the activity of VL to bind NH-LBP was determined by ELISA.Results The results showed that the cysteine was introduced into the position 21 amino acid of VL to replace the threonine.The gene of VL was about 650 bp and relative molecular weight of VL was 28?103.VL could bind NH-LBP directly.Conclusion These have laid a foundation for producing the dsFv against NH-LBP.