Application of molecular probes in nuclear imaging of neuroendocrine tumors / 浙江大学学报·医学版

Neuroendocrine tumors are a type of heterogeneous tumors originating from neuroendocrine cells derived from the neural crest，which can secrete a variety of amines and peptide hormones.Based on different molecular biomarkers，histologic types and differentiation degrees，individualized nuclear imaging can provide information for the early diagnosis，clinical staging，treatment guidance，and detection of the recurrence and metastasis of neuroendocrine tumor. In this paper，we review the development and application of nuclear medicine molecular imaging probes such as glucose analogs，somatostatin analogues，amine precursors，hormone analogs and enzyme inhibitors in the diagnosis and treatment of neuroendocrine tumors.

Improved synthesis process of optical-magnetic bimodal probe of Gd-4, 7-Bis-carboxymethyl-10-(2-fluoresceinthioureaethyl)-1, 4, 7, 10-tetraaza-cyclododec-1-yl-acetic acid complexes / 北京大学学报(医学版)

OBJECTIVE@#To improve the methods to synthesize and purify of optical-magnetic bimodal molecular probe of Gd-[4, 7-Bis-carboxymethyl-10-(2-fluorescein thioureaethyl)-1, 4, 7, 10-tetraaza-cyclododec-1-yl]-acetic acid complexes.@*METHODS@#Target compound (7), optical-magnetic bimodal molecular molecular probe, was synthesized by the use of 1, 4, 7, 10-tetraazacyclododecane (1) as starting material via substitution reaction, hydrolysis reaction, coupling reaction and complexation reaction with metal.@*RESULTS@#The synthetic route of Gd-[4, 7-Bis-carboxymethyl-10-(2-fluoresceinthioureaethyl)-1, 4, 7, 10-tetraaza-cyclododec-1-yl]-acetic acid complexes was improved. The optical-magnetic bimodal molecular probes were synthesized by substitution reaction, hydrolysis reaction, coupling reaction and complex reaction with metal respectively. For the improved route, the total yield could reach 34.6% which was higher than the original route (18.0%). The structures of those compounds were identified by 1H nuclear magnetic resonance, 13C nuclear magnetic resonance, and mass spectrometry. The improved route could avoid the uncontrollable disadvantage of the substitution reaction, this process could reduce the formation of impurities and made the purification process easier, and in the aspect of purification and separation, the preparative high-performance liquid chromatography with less sample loading and high cost was improved to a column chromatography with many sample loads and being easy to operate. Therefore, the use of column chromatography could be more conducive to mass production of the optical-magnetic bimodal molecular molecular probe.@*CONCLUSION@#The improved synthetic route improves the controllability of the reaction conditions and makes it easier to purify and separate the compounds. At the same time, the improved synthetic route can increase the total yield significantly. The optical-magnetic bimodal molecular probe can combine the living magnetic resonance imaging with the in vitro optical imaging to realize the dual synchronous detection of magneto-optics, so that the detection results of the living magnetic resonance imaging and the in vitro optical imaging are mutually verified. In other words, this synthetic optical-magnetic bimodal molecular probe will make the experimental results more accurate and reliable. In subsequent biological experimental studies, the optical-magnetic bimodal molecular probe can be applied to related research of brain structure and function, and the probe can be used for the brain-related diseases researches, such as brain tumors. after intravenous administration, and thus the optical-magnetic bimodal molecular probe can play an important role in medical treatment of brain tumors and cerebrovascular diseases.

Application of anoptomagnetic probe Gd-DO3A-EA-FITC in imaging and analyzing the brain interstitial space / 北京大学学报(医学版)

OBJECTIVE@#To investigate the application of the optical magnetic bimodal molecular probe Gd-DO3A-ethylthiouret-fluorescein isothiocyanate (Gd -DO3A-EA-FITC) in brain tissue imaging and brain interstitial space (ISS).@*METHODS@#In the study, 24 male SD rats were randomly divided into 3 groups, including magnetic probe group (n=6), optical probe group (n=6) and optical magnetic bimodal probe group (n=12), then the optical magnetic bimodal probe group was divided equally into magnetic probe subgroup (n=6) and optical probe subgroup (n=6). Referencing the brain stereotaxic atlas, the coronal globus pallidus as center level, the probes including gadolinium-diethylene triamine pentaacetic acid (Gd-DTPA), fluorescein isothiocyanate (FITC) and Gd-DO3A-EA-FITC of 2 μL (10 mmol/L) were injected into the caudate nucleus respectively, magnetic resonance imaging (MRI) was performed in the magnetic probe group and magnetic probe subgroup to image the dynamic diffusion and distribution of the probes in the brain ISS, a self-developed brain ISS image processing system was used to measure the diffusion coefficient, clearance, volume fraction and half-time in these two groups. Laser scanning confocal microscope (LSCM) was performed in vitro in the optical probe group and optical probe subgroup for fluorescence imaging at the time points 2 hours after the injection of the probe, and the distribution in the oblique sagittal slice was compared with the result of the first two groups.@*RESULTS@#For the magnetic probe group and magnetic probe subgroup, there were the same imaging results between the probes of Gd-DTPA and Gd-DO3A-EA-FITC. The diffusion parameters of Gd-DTPA and Gd-DO3A-EA-FITC were as follows: the average diffusion coefficients [(3.31±0.11)×10-4 mm2/s vs. (3.37±0.15)×10-4 mm2/s, t=0.942, P=0.360], the clearance [(3.04±0.37) mmol/L vs. (2.90±0.51) mmol/L, t=0.640, P=0.531], the volume fractions (17.18%±0.14% vs. 17.31%±0.15%, t=1.961, P=0.068), the half-time [(86.58±3.31) min vs. (84.61±2.38) min, t=1.412, P=0.177], the diffusion areas [(23.25±0.68) mm2 vs. (22.71±1.00) mm2, t=1.100, P=0.297]. The statistical analysis of each brain was made by t test, and the diffusion parameters were not statistically significant. Moreover, for the optical probe group and optical probe subgroup, the diffusion area of Gd-DO3A-EA-FITC [(22.61±1.16) mm2] was slightly larger than that of FITC [(22.10±1.29) mm2], the statistical analysis of each brain was made by t test, and the diffusion parameters were not statistically significant (t=0.713, P=0.492).@*CONCLUSION@#Gd-DO3A-EA-FITC shows the same imaging results as the traditional GD-DTPA, and it can be used in measuring brain ISS.

Review: optically-triggered phase-transition droplets for photoacoustic imaging

Optically-triggered phase-transition droplets have been introduced as a promising contrast agent for photoacoustic and ultrasound imaging that not only provide significantly enhanced contrast but also have potential as photoacoustic theranostic molecular probes incorporated with targeting molecules and therapeutics. For further understanding the dynamics of optical droplet vaporization process, an innovative, methodical analysis by concurrent acoustical and ultrafast optical recordings, comparing with a theoretical model has been employed. In addition, the repeatability of the droplet vaporization-recondensation process, which enables continuous photoacoustic imaging has been studied through the same approach. Further understanding the underlying physics of the optical droplet vaporization and associated dynamics may guide the optimal design of the droplets. Some innovative approaches in preclinical studies have been recently demonstrated, including sono-photoacoustic imaging, dual-modality of photoacoustic and ultrasound imaging, and super-resolution photoacoustic imaging. In this review, current development of optically triggered phase-transition droplets and understanding on the vaporization dynamics, their applications are introduced and future directions are discussed.

Fontes de obtenção de medicamentos para tratamento de hipertensão arterial no Brasil: análise da Pesquisa Nacional de Saúde, 2013 / Sources for obtaining drugs for hypertension in Brazil: results from the National Health Survey, 2013

RESUMO: Objetivo: Analisar as diferenças sociodemográficas dos indivíduos adultos hipertensos, em relação às fontes de obtenção de medicamentos para tratar hipertensão arterial no Brasil. Métodos: Análise secundária dos dados oriundos da Pesquisa Nacional de Saúde, 2013; os desfechos considerados nas análises foram representados pelas fontes de obtenção de medicamentos para tratar a hipertensão arterial. Resultados: Foram entrevistados 10.017 indivíduos. A grande maioria dos hipertensos em uso de medicamentos (74,0%) utiliza uma fonte única de obtenção de medicamentos 7,3% (IC95% 6,4 - 8,4) referiu obter todos os medicamentos por meio dos planos de saúde privados; 22,7% (IC95% 21,0 - 24,4) em farmácias do sistema público de saúde; 21,8% (IC95% 20,2 - 23,4) no Programa Farmácia Popular do Brasil; e 29,5% (IC95% 27,7 - 31,4) exclusivamente pelas farmácias comerciais. A obtenção no sistema público de saúde como fonte única diminuiu com o avanço da idade, apresentou-se menor nas pessoas de cor da pele branca, diminuiu fortemente com o aumento da escolaridade e evidenciou-se menor entre os residentes na região Norte do país; no Programa Farmácia Popular do Brasil, a fonte única de obtenção também foi menor para as pessoas com maior escolaridade. A obtenção nas farmácias comerciais esteve associada positivamente com um perfil do sexo masculino, de maior escolaridade, de idade mais elevada, tendo declarado cor da pele branca. A ocorrência de mais de uma fonte de obtenção mostrou-se associada positivamente ao aumento da idade e inversamente ao aumento da escolaridade. Conclusões: Os resultados possibilitaram identificar diferentes estratégias para obtenção de medicamentos usados no tratamento da hipertensão, de modo a explicar como são obtidos os medicamentos no país e qual o impacto das políticas públicas nesse setor.

ABSTRACT: Objective: To analyze the sociodemographic differences among adults with hypertension regarding the sources for obtaining drugs for hypertension treatment in Brazil. Methods: This is a secondary analysis of data from the National Health Survey 2013; the outcomes considered for the analysis were the sources for obtaining drugs for treating high blood pressure. Results: The great majority (74%) of patients with hypertension taking drugs use a single source for obtaining them, 7.3% (95%CI 6.4 - 8.4) reported getting all the drugs through private health plans, 22.7% (95%CI 21.0 - 24.4) by pharmacies of the public health system, 21.8% (95%CI 20.2 - 23.4) by the Popular Pharmacy Program, and about one-third (29.5%; 95%CI 27.7 - 31.4) exclusively by commercial pharmacies. Having the public health system as the single source for obtaining the drugs was found to decrease with age, was lower in white people, decreased strongly with increase in education, and was lower for residents in the North region. Exclusive obtainment through the Popular Pharmacy Program was lower for people with higher education. Obtainment in commercial pharmacies was positively associated with being male, with higher education level, being older, and having white skin color. Obtainment using more than one source was positively associated with increasing age and inversely associated with higher education levels. Conclusions: The results allowed the identification of a trajectory of patients in obtaining drugs for the treatment of hypertension, aiming at explaining how the drugs are obtained and the impact of public policies in this sector in the country.

Advances in the Endoscopic Assessment of Inflammatory Bowel Diseases: Cooperation between Endoscopic and Pathologic Evaluations

Endoscopic assessment has a crucial role in the management of inflammatory bowel disease (IBD). It is particularly useful for the assessment of IBD disease extension, severity, and neoplasia surveillance. Recent advances in endoscopic imaging techniques have been revolutionized over the past decades, progressing from conventional white light endoscopy to novel endoscopic techniques using molecular probes or electronic filter technologies. These new technologies allow for visualization of the mucosa in detail and monitor for inflammation/dysplasia at the cellular or sub-cellular level. These techniques may enable us to alter the IBD surveillance paradigm from four quadrant random biopsy to targeted biopsy and diagnosis. High definition endoscopy and dye-based chromoendoscopy can improve the detection rate of dysplasia and evaluate inflammatory changes with better visualization. Dye-less chromoendoscopy, including narrow band imaging, iScan, and autofluorescence imaging can also enhance surveillance in comparison to white light endoscopy with optical or electronic filter technologies. Moreover, confocal laser endomicroscopy or endocytoscopy have can achieve real-time histology evaluation in vivo and have greater accuracy in comparison with histology. These new technologies could be combined with standard endoscopy or further histologic confirmation in patients with IBD. This review offers an evidence-based overview of new endoscopic techniques in patients with IBD.

Effects of external magnetic field on the transfection rate of SPIO-shRNADual functional molecular probe into ovarian carcinoma SKOV3 cells in vitro / 中国医学科学院学报

<p><b>OBJECTIVE</b>To explore the transfection rate of SPIO-shRNA dual functional molecular probe into ovarian carcinoma SKOV3 cells in external magnetic field.</p><p><b>METHODS</b>Dual functional molecular probe at an iron concentration of 45 mg/L was transfected into SKOV3 cells. The cells with coexisting probe and magnetic fields were set as the intervention group,the probe-transfected cells as negative control group, and normally cultured SKOV3 without any transfection as blank control group. The transfection rate was detected by flow cytometry. Cell viability was observed by CCK-8 assay. Epidermal growth factor receptor (EGFR) expression level in SKOV3 cells was determined by real-time quantitative PCR and Western blot analysis. The signal intensity was measured by magnetic resonance imaging (MRI).</p><p><b>RESULTS</b>The transfection rate of the intervention group was (79.20 ± 3.31)%, which was significantly higher than that of negative control group (P=0.001). Compared with the negative control group,the cell viability of the intervention group significantly decreased (P=0.011), protein and mRNA expression levels of EGFR in the intervention group were significantly decreased (both P<0.05). The signal intensity on T2(*)WI in the intervention group also significantly decreased (P=0.0004).</p><p><b>CONCLUSION</b>The external magnetic field can improve the transfection efficiency SPIO-shRNA dual functional molecular probe into ovarian carcinoma SKOV3 cells.</p>

Pharmacokinetics and MR imaging of SPIO-shRNA dual functional molecular probe in vivo / 药学学报

In this study, we investigated the pharmacokinetics parameters of SPIO-shRNA dual functional molecular probe and observed the main organ distribution by MRI in vivo. Eighteen New Zealand white rabbits were randomly divided into three groups and injected intravenously with different doses of SPIO-shRNA molecular probe, respectively. The blood samples were collected to analyze the pharmacokinetic parameters by measuring the iron content at 30 minutes before and after the injection. Twenty-four Kun Ming (KM) mice were randomly divided into 4 groups: the control group was injected intravenously with physiological saline 200 µL per mouse via the tail vein, the other 3 groups were injected intravenously with different doses of SPIO-shRNA molecular probe. MRI observation was performed in 24 hours, and the liver, spleen, kidney, brain and muscle were collected for iron quantification with Prussian blue staining to determine distribution of the SPIO-shRNA molecular probe in the main organ in vivo. Our results suggest that the molecular probe blood half-life is more than 3 hours. The data of MRI suggest the probe was distributed in liver and spleen, and the MRI signal was reduced with the increase in probe's doses (P < 0.05). The results of Prussian blue staining confirmed the results of MRI. Most of the probe could escape the phagocytosis of mononuclear phagocyte system. Our data provide the pharmacokinetic and distribution of SPIO-shRNA molecular probe in organs. Meanwhile, it suggests the choice of the time and dose of probe for MR imaging of tumor in vivo.

Nanobody: a Novel Molecular Imaging Tool / 中国医疗器械杂志

Nanobodies are derived from the variable domain of the heavy-chain antibodies (HCAbs) that occur naturally in the serum of camels. Using nanobody-based probes, several imaging techniques such as radionuclide-based, optical and ultrasound have been employed for visualization of target expression in various disease models. Combined with application and clinical data of nanobody in molecular imaging in recent years, this paper introduces its application in the diagnosis of diseases and the future development as a novel molecular imaging tool.

Progress in the study of near-infrared fluorescent probes for the detection of β-amyloid deposition in Alzheimer's disease / 药学学报

Alzheimer's disease (AD) is the most common cause of cognitive impairment in older people. With the aging of society is more and more serious, AD caused great burden to patients and society. A β is a classical biomarker of AD, which has been widely used in clinical diagnosis of AD patients. Compared with positron emission tomography (PET) and single photon emission computed tomography (SPECT), near infrared fluorescence imaging has many advantages including highly sensitive, non-invasive, safety and inexpensive. Therefore, many research groups have focused on developing the molecular probes of near-infrared fluorescence (NIRF) imaging. In this article, we will review the progress of the probes of NIRF.

Robertsonian chromosomes and the nuclear architecture of mouse meiotic prophase spermatocytes

BACKGROUND: The nuclear architecture of meiotic prophase spermatocytes is based on higher-order patterns of spatial associations among chromosomal domains from different bivalents. The meiotic nuclear architecture depends on the chromosome characteristics and consequently is prone to modification by chromosomal rearrangements. In this work, we consider Mus domesticus spermatocytes with diploid chromosome number 2n = 40, all telocentric, and investigate a possible modification of the ancestral nuclear architecture due to the emergence of derived Rb chromosomes, which may be present in the homozygous or heterozygous condition. RESULTS: In the 2n = 40 spermatocyte nuclei random associations mediated by pericentromeric heterochromatin among the 19 telocentric bivalents ocurr at the nuclear periphery. The observed frequency of associations among them, made distinguishable by specific probes and FISH, seems to be the same for pairs that may or may not form Rb chromosomes. In the homozygote Rb 2n = 24 spermatocytes, associations also mediated by pericentromeric heterochromatin occur mainly between the three telocentric or the eight metacentric bivalents themselves. In heterozygote Rb 2n = 32 spermatocytes all heterochromatin is localized at the nuclear periphery, yet associations are mainly observed among the three telocentric bivalents and between the asynaptic axes of the trivalents. CONCLUSIONS: The Rb chromosomes pose sharp restrictions for interactions in the 2n = 24 and 2n = 32 spermatocytes, as compared to the ample possibilities for interactions between bivalents in the 2n = 40 spermatocytes. Undoubtedly the emergence of Rb chromosomes changes the ancestral nuclear architecture of 2n = 40 spermatocytes since they establish new types of interactions among chromosomal domains, particularly through centromeric and heterochromatic regions at the nuclear periphery among telocentric and at the nuclear center among Rb metacentric ones.

The Effect of RGP Lens and Reverse Geometry Lens on Redistribution of Corneal Epithelial Cell in Rabbit

PURPOSE: To investigate the migration and redistribution of rabbit corneal epithelial cells when wearing reverse geometry lens (RGL) or rigid gas permeable lens (RGP). METHODS: In 30 rabbits, the right eyes were fitted with either RGL or RGP and the left eyes were untreated to serve as controls. The rabbits were sacrificed at 1, 3, 7, 10 and 14 days after lens fitting. The central and peripheral corneal thicknesses were measured by microscope and the ratio of right to left corneal thickness was calculated to evaluate the characteristics of change over time. By using the molecular probe 7-nitrobenz-2-ox-1,3-diazolylphallacidin (NBD phallacidin), the samples were examined with light microscope to determine the migration and redistribution of epithelial cells in the rabbit cornea. RESULTS: No consistent changes in the thickness of both central and peripheral corneal epithelium were found. The corneal epithelial cells of both eyes with RGL and RGP reacted positively to NBD phallacidin. The fluorescence was most increased at day 3 of sacrifice in RGL cases and at day 7 in RGP cases, and then decreased in both cases. The corneal epithelium of eyes with RGL exhibited marked increase in the intensity of fluorescence compared to the eyes with RGP. CONCLUSIONS: The corneal epithelium with RGL showed the strongest intensity of NBD phallacidin fluorescence. This result suggests that wearing RGL may induce the migration and redistribution of corneal epithelial cells.

Optical Molecular Imaging for Diagnosing Intestinal Diseases

Real-time visualization of the molecular signature of cells can be achieved with advanced targeted imaging techniques using molecular probes and fluorescence endoscopy. This molecular optical imaging in gastrointestinal endoscopy is promising for improving the detection of neoplastic lesions, their characterization for patient stratification, and the assessment of their response to molecular targeted therapy and radiotherapy. In inflammatory bowel disease, this method can be used to detect dysplasia in the presence of background inflammation and to visualize inflammatory molecular targets for assessing disease severity and prognosis. Several preclinical and clinical trials have applied this method in endoscopy; however, this field has just started to evolve. Hence, many problems have yet to be solved to enable the clinical application of this novel method.

Progress of research on application of quantum dots in tissue engineering / 生物医学工程学杂志

Quantum dots (QDs) are novel photo-stable semiconductor nanocrystals with wide excitation spectra and narrow, symmetrical emission spectra. QDs can be used as molecular probes by conjugating to a wide range of biological targets, including proteins, peptides and nucleic acids. It has been widely used in bio-labeling, fast detection and biological imaging. In this review, we focus on the applications of QDs in tissue engineering and its potential bio-safety issues.

(99m)Tc radiolabeling of a novel polypeptide molecular probe for lung cancer and its biodistribution in animals / 南方医科大学学报

<p><b>OBJECTIVE</b>To develop a method for (99m)Tc radiolabeling of a small molecular peptide targeting lung carcinoma and observe the biokinetics and biodistribution of the labeled peptide in normal mice and rabbits.</p><p><b>METHODS</b>MAG3-peptide (cNGQGEQc) was labeled with (99m)Tc and the labeling rate was determined with paper chromatography. In vitro stability test, cysteine challenge test and serum incubation test were performed for radiochemical evaluation of the labeled peptide. Blood (99m)Tc-peptide clearance in rabbits was evaluated by determining blood radioactive concentrations at different time points after injection of 37 MBq (99m)Tc-peptide, and its dynamic distribution was investigated by SPECT imaging. The percent injected dose per gram of tissue was calculated for each organ of mice injected intravenously with 7.4 MBq (99m)Tc-peptide based on gamma counter readings.</p><p><b>RESULTS</b>The labeling rate of (99m)Tc-peptide exceeded 90%, and the radiochemical purity was 91% after placing for 12 h at room temperature and 85% after incubation at 37 degrees celsius; with human serum. The cysteine replacement rate was less than 7%, and the binding rate of (99m)Tc-peptide with serum proteins was below 5%. SPECT imaging showed that the labeled peptide could be quickly cleared from the blood in normal animals primarily through the kidneys, and the radioactivity in other tissues and organs remained low.</p><p><b>CONCLUSION</b>(99m)Tc-peptide can be easily prepared with a high labeling yield. With good stability both in vitro and in vivo, (99m)Tc-peptide can be quickly cleared from the blood and excreted though the kidney with ideal biodistribution and biokinetics in vivo.</p>

The application of small molecule bioactive probes in the identification of cellular targets / 药学学报

Identification of the cellular targets of bioactive compounds is a major challenge and a key issue in chemical biology and drug discovery. As an important technology in functional proteomics, small molecule probes play a pivotal role in the identification of cellular targets of bioactive compounds. This review is intended to introduce the application principles and structural design philosophy of chemical probes for the purpose of mechanistic study. Recent cases of successful application were also discussed to further demonstrate the principles and significance ofbioactive small molecule-based probes.

Research progress of aptamers application in molecular imaging / 生物医学工程学杂志

Molecular imaging technology, an advanced research area of imaging, can provide real-time, non-invasive image information of the target site in molecular level. The key of the molecular imaging technology is molecular probe. Aptamers are short oligonucleotides with high affinity and specificity to the target molecules. The targeting ability, stability and safety of aptamers are superior to traditional antibodies so that aptamers show prosperous usages in targeting drug delivery and disease diagnostics. Therefore, aptamers are considered to be an extremely ideal probes, which can guide quantum dots, magnetic nanoparticles and ultrasound contrast agents on the targets and realize optical, magnetic resonance, ultrasonic multimodal and multifunctional imaging. All of the advantages can further promote the application of molecular imaging in disease treatment and diagnosis. In this paper, we review recent developments in the application of aptamers as molecular probes in major branches of molecular imaging.

Incorporación química de la sonda fluorescente FlAsH en la proteína Hha: aplicación al estudio del complejo Hha/H-NS / Chemical incorporation of fluorescent probe flAsH on the Hha protein: application to the study of complex Hha/H-NS

El objetivo de esta investigación fue realizar estudios de incorporación de la sonda FlAsH como posible herramienta para el estudio in vitro e in vivo de la proteína Hha y de su interacción con H-NS. Se construyó una proteína con la secuencia específica “CCPGCC” capaz de unir la sonda fluorescente FlAsH; posteriormente se desarrollaron procesos de transformación, expresión y purificación, con los cuales se evidenció que a pesar de introducir una secuencia adicional no nativa a la proteína, se pudo obtener proteína en buena cantidad, estabilidad, rendimiento y con un alto nivel de pureza. La optimización del protocolo de incorporación del FlAsH se hizo teniendo en cuenta el rendimiento y el tiempo de reacción. El complejo FlAsH/HhaCCPGCC se caracterizó mediante fluorescencia y se comprobó mediante MALDI TOF. La incorporación HhaCCPGCC1/ FlAsH no se obtuvo en un alto porcentaje como se esperaba, por esta razón no se pudieron hacer los estudios de interacción entre el complejo de proteínas Hha y H-NS.

Evaluation of alpha-Tubulin as an Antigenic and Molecular Probe to Detect Giardia lamblia

The alpha/beta-tubulin heterodimer is the basic subunit of microtubules in eukaryotes. Polyclonal antibodies specific to recombinant alpha-tubulin of Giardia lamblia were made, and found effective as a probe to specifically detect G. lamblia by immunofluorescence assays. Nucleotide sequences of alpha-tubulin genes were compared between G. lamblia WB and GS strains, prototypes of assemblage A and assemblage B, respectively. A set of primers was designed and used to amplify a portion of the alpha-tubulin gene from G. lamblia. PCR-RFLP analysis of this alpha-tubulin PCR product successfully differentiated G. lamblia into 2 distinct groups, assemblages A and B. The results indicate that alpha-tubulin can be used as a molecular probe to detect G. lamblia.

Quantitation of HTLV-I proviral load using real-time quantitative PCR with Taqman MGB probe / 病毒学报

A quantitative real-time PCR assay was developed to measure the proviral load of human T-lymphotropic virus type I (HTLV-I) in peripheral blood. The technology utilizes special primers and Taqman MGB fluorescence probe to measure amplification products from the gag-pro-pol polyprotein gene of HTLV-I. HTLV-I copy number was normalized to the amount of cellular DNA by quantitation of the beta-actin gene, The amplification system was sensitive to detect 5 copy/microL. The standard curve had a good linearity when the quantity for the gene was between 10(3) and 10(7) copy/microL (R2 = 0.999). Good reproducibility was observed in each intra- and inter-assay. We also measured proviral load in peripheral blood in 12 HTLV-I seropositive former blood donors. Proviral load for HTLV-I infected donors ranged from 0.015 to 12.819 copy/cell in WBC with the mean of 3.116 copy/cell.