Missed opportunities for screening congenital syphilis early during pregnancy: A case report and brief literature review.

Congenital syphilis is a significant public health problem. Pregnant women infected with Treponema pallidum present with various clinical manifestations, mainly including skin or visceral manifestations. The extensive clinical manifestations of T. pallidum infection mimic those of many other diseases during pregnancy, which may lead to delayed diagnosis and serious consequences. We report a case of fetal T. pallidum infection and premature delivery in a woman whose syphilis screening was negative at 16 weeks of gestation. Despite presenting to the dermatologist at 24 weeks of gestation with maculopapular rash which is usually associated with secondary syphilis, the diagnosis of syphilis was not considered. This case shows that even if early syphilis screening of pregnant women is negative, they may still get infected with T. pallidum later on in pregnancy. Therefore, in patients presenting with a rash without an obvious cause, T. pallidum infection should be excluded. The health status of patients' spouses should be assessed during pregnancy. Additionally, perinatal health education is necessary for women and their spouses during pregnancy. The abovementioned factors could reduce the probability of T. pallidum infection in pregnant women and their infants.

A Novel PCR Method for Detecting ACE Gene Insertion/Deletion Polymorphisms and its Clinical Application.

BACKGROUND: Angiotensin-converting enzyme (ACE) plays a major role in blood pressure regulation and cardiovascular homeostasis. The wide distribution and multifunctional properties of ACE suggest it's involvement in various pathophysiological conditions. RESULTS: In this study, a novel visual detection method for ACE I/D polymorphisms was designed by integrating direct PCR without the need for DNA extraction using gold magnetic nanoparticles (GMNPs)-based lateral flow assay (LFA) biosensor. The entire detection procedure could enable the genotyping of clinical samples in about 80 min. The detection limit was 0.75 ng and results could be obtained in 5 min using the LFA device. Three hundred peripheral blood samples were analyzed using the direct PCR-LFA system and then verified by sequencing to determine accuracy and repeatability. A clinical preliminary study was then performed to analyze a total of 633 clinical samples. CONCLUSIONS: After grouping based on age, we found a significant difference between the genotypes and the age of patients in the CHD group. The introduction of this method into clinical practice may be helpful for the diagnosis of diseases caused by large fragment gene insertions/deletions.

Anti-Hypertensive Action of Fenofibrate via UCP2 Upregulation Mediated by PPAR Activation in Baroreflex Afferent Pathway.

Fenofibrate, an agonist for peroxisome proliferator-activated receptor alpha (PPAR-α), lowers blood pressure, but whether this action is mediated via baroreflex afferents has not been elucidated. In this study, the distribution of PPAR-α and PPAR-γ was assessed in the nodose ganglion (NG) and the nucleus of the solitary tract (NTS). Hypertension induced by drinking high fructose (HFD) was reduced, along with complete restoration of impaired baroreceptor sensitivity, by chronic treatment with fenofibrate. The molecular data also showed that both PPAR-α and PPAR-γ were dramatically up-regulated in the NG and NTS of the HFD group. Expression of the downstream signaling molecule of PPAR-α, the mitochondrial uncoupling protein 2 (UCP2), was up-regulated in the baroreflex afferent pathway under similar experimental conditions, along with amelioration of reduced superoxide dismutase activity and increased superoxide in HFD rats. These results suggest that chronic treatment with fenofibrate plays a crucial role in the neural control of blood pressure by improving baroreflex afferent function due at least partially to PPAR-mediated up-regulation of UCP2 expression and reduction of oxidative stress.

Treatment of Graves' ophthalmopathy with an in-house Phosphorus-32 source: Initial clinical observations.

The objective of the present study was to observe the therapeutic effect of radiation delivered via a 32P source on Graves' ophthalmopathy. A32P solution was injected into a 10-ml vacuum flask held inside a lead container. A window was cut in the lead, generating a treatment beam. Radiation was given to four areas: The upper and lower orbit (covering ~1/3 of the eyelid) and the inner and outer canthus. Each site received 10 daily doses of 20 cGy. Proptosis was measured by an exophthalmometer and the palpebral aperture was determined with a ruler. Measurements were taken before and after the treatment. After 5 days of treatment, the patient displayed a significant improvement, and by 10 days, the average reduction of proptosis in Graves' ophthalmopathy was 3.36±1.73 mm for the left and 3.05±2.04 mm for the right eyes. The treatment was effective in all patients, who uniformly reported rapid pain relief. Conjunctival congestion and eyelid edema also improved significantly. However, only 50% of patients showed improved diplopia after treatment, which was poor compared with other symptoms. No obvious side effects were found in the subsequent follow-up. In conclusion, 32P brachytherapy for Graves' ophthalmopathy was simple and effective, with few side effects, and should be considered as a promising therapy.

Sensitive genotyping of mutations in the EGFR gene from NSCLC patients using PCR-GoldMag lateral flow device.

Epidermal growth factor receptor (EGFR) mutations predict better outcomes with EGFR tyrosine kinase inhibitors in patients with non-small cell lung cancer (NSCLC). Most common activating mutations include in-frame deletion in exon 19 and L858R substitution in exon 21, which account for >90% of all EGFR mutations in NSCLC. In this study, a PCR-GoldMag lateral flow assay (PCR-GoldMag LFA) was developed for the visual detection of delE746-A750 and L858R of EGFR mutations. Forty formalin-fixed paraffin-embedded (FFPE) tissue samples of NSCLC patients were analyzed using PCR-GoldMag LFA system and verified by direct sequencing and TaqMan-PCR detection methods. Results showed that EGFR mutations were detected in 34 cases among the 40 samples (85%) by PCR-GoldMag LFA method. Among the 34 cases, 5 cases were simultaneously detected with delE746-A750 in exon 19 and L858R mutation in exon 21. Compared with sequencing, only 4 samples were detected as delE746-A750, which revealed higher sensitivity of PCR-GoldMag LFA detection method than direct sequencing. TaqMan-PCR method verified the L858R mutation and was in 100% agreement with our method. These results indicated that our method has obvious advantages to analyze clinical samples and offers a more sensitive alternative to direct sequencing for the detection of EGFR mutations.

[Immunoprotective effect of combined pneumococcal endopeptidase O and pneumococcal surface adhesin A vaccines against Streptococcus pneumoniae infection].

OBJECTIVE: To investigate the prokaryotic expression of proteins pneumococcal endopeptidase O (PepO) and pneumococcal surface adhesin A (PsaA) in Streptococcus pneumoniae and their immunoprotective effect as vaccine candidate proteins. METHODS: Specific primers of target gene fragments were designed, and then PCR amplification was performed to establish recombinant plasmids pET28a(+)-pepO and pET28a(+)-psaA, which were transformed into host cells, Escherichia coli BL21 and DE3, respectively, to induce expression. Highly purified target proteins PepO and PsaA were obtained after purification. Mucosal immunization was performed for BALB/c mice and specific antiserum was prepared. ELISA was used to measure the antibody titer, and Western blot was used to analyze the specificity of the antiserum of target proteins. The mice were randomly divided into negative control group, PepO group, PsaA group, and PepO+PsaA combined immunization group, with 18 mice in each group. The models of different serotypes of Streptococcus pneumoniae infection were established to evaluate the immunoprotective effect of target proteins used alone or in combination. RESULTS: The target proteins PepO and PsaA were successfully obtained and Western blot demonstrated that the antiserum of these proteins had good specificity. There was no significant difference in the titers of IgA in saliva and IgG in serum between the PepO group and the combined immunization group (P>0.05); however, these two groups had significantly higher antibody titers than the PsaA group (P<0.05). The PepO, PsaA, and combined immunization groups had significantly higher protection rates for mice infected with Streptococcus pneumoniae D39 and CMCC31436 in the nasal cavity than the negative control group (P<0.05). The PepO and combined immunization groups had a significantly higher protection rate for mice infected with Streptococcus pneumoniae D39 than the PsaA group (P<0.05). The results of colonization experiment showed that compared with the control group, the PepO, PsaA, and combined immunization groups showed a significant reduction in the colonization of Streptococcus pneumoniae (CMCC31693 and CMCC31207) in the nasopharynx and lung (P<0.05). The combined immunization group showed a better effect on reducing the colonization of CMCC31207 in the lung than the PepO and PsaA alone groups. CONCLUSIONS: Combined PepO/PsaA vaccines may produce a better protective effect by mucosal immunization compared with the vaccine used alone in mice. The combined vaccines can effectively reduce the colonization of Streptococcus pneumoniae in the nasopharynx and lung. Therefore, such protein vaccines may have a great potential for research and development.

Establishment and application of a real-time loop-mediated isothermal amplification system for the detection of CYP2C19 polymorphisms.

Single-nucleotide polymorphisms (SNPs) represent the most widespread type of genetic variation (approximately 90%) in the human genome, and the demand to overcome such variation has received more attention now than ever before. The capacity to rapidly assess SNPs that correlate with disease predisposition, drug efficacy and drug toxicity is a key step for the development of personalized medicine. In this work, a rapid one-step SNP detection method, real-time loop-mediated isothermal amplification (RT-LAMP), was first applied for CYP2C19 polymorphisms testing. The optimized method was established with specifically designed primers for target amplification by real-time detection in approximately 30 min under isothermal conditions. RT-LAMP amplified few copies of template to produce significant amounts of product and quantitatively detected human DNA with compatible specificity and sensitivity. The success in the establishment of this RT-LAMP protocol for CYP2C19 polymorphism testing is significant for the extension of this technique for the detection of other SNPs, which will further facilitate the development of personalized medicine.

(18)F-fluoromisonidazole PET reveals spatial and temporal heterogeneity of hypoxia in mouse models of human non-small-cell lung cancer.

AIM: To noninvasively observe dynamic changes in tumor hypoxia in mouse models of human non-small-cell lung cancer (NSCLC) using (18)F-fluoromisonidazole PET. MATERIALS & METHODS: Nude mice with NSCLC H460 and A549 subcutaneous xenografts were coinjected intravenously with (18)F-fluoromisonidazole and the hypoxia marker pimonidazole, and observed by serial PET scans. After sacrifice, the tumor distribution of (18)F-fluoromisonidazole and pimonidazole was compared by digital autoradiography and microscopy, respectively. RESULTS: The NSCLC hypoxic microenvironment was spatially heterogeneous. Serial PET scans over 48 h revealed an apparent change in the intratumoral distribution of (18)F-fluoromisonidazole. CONCLUSION: The tumor hypoxic microenvironment is spatially and temporally heterogeneous, and hypoxic cancer cells have a shorter life span when growing in vivo. Therefore, the concept of hypoxic resistance and hypoxia-targeting therapy of macroscopic tumors should be revisited.

Polymorphisms and phenotypic analysis of cytochrome P450 2D6 in the Tibetan population.

The cytochrome P450 2D6 enzyme (CYP2D6) metabolizes about 25% of prescribed drugs in the endoplasmic reticulum, and genetic polymorphisms in CYP2D6 can greatly affect its activity and lead to differences among individuals in drug efficacy and adverse drug reactions. To investigate genetic polymorphisms in CYP2D6 among Tibetan Chinese, we directly sequenced the whole gene in 96 unrelated, healthy Tibetans from The Tibet Autonomous Region of China and screened for genetic variants in the promoter, exons, introns, and 3'UTR. We detected fifty-one genetic polymorphisms in CYP2D6, and 16 of them are novel. The allele frequencies of CYP2D6*1, *2, *5, *10, *41, and *49 were 0.25, 0.43, 0.02, 0.29, 0.02, and 0.01, respectively. The frequency of CYP2D6*10, a putative poor-metabolizer allele, was lower in our sample population compared with that in the Han Chinese population (p<0.001). In addition, haplotype analysis allowed 15 CYP2D6 haplotypes to be classified into three groups. In conclusion, our results provide basic information about CPY2D6 alleles in Tibetans and suggest that the enzymatic activities of CYP2D6 may differ among the diverse ethnic populations of China. Our results provide a basis for safer drug administration and better therapeutic treatment among Tibetans.

[Amino-terminal pro-brain natriuretic peptide and brain natriuretic peptide measurements under various detection conditions in patients with chronic heart failure].

OBJECTIVE: To find the potential interference factors for the detection of NT-proBNP and BNP in patients with chronic heart failure. METHODS: EP15-A2 issued by Clinical and Laboratory Standards Institute (CLSI) was employed to compare the precision and accuracy of commercial NT-proBNP and BNP analyzer electrochemiluminescence immunoassay system Cobas E601 and chemiluminescence system ADVIA Centaur. Moreover, NT-proBNP and BNP were detected in different time interval and in different interfered sampling conditions (haematolysis, choloplania, lipemia). NT-proBNP and BNP of 203 patients with heart failure or heart failure complicated with acute cerebral infarction were analyzed to find the deviation caused by patients' endogenous factors. RESULTS: The precision and accuracy were comparable for NT-proBNP and BNP detection using Cobas E601 and ADVIA Centaur (total-CV below 2.9% and 3.5%, the deviation from definite value below 2.38% and 3.91%). The most suitable sample type for NT-proBNP and BNP detection was serum and EDTA-anticoagulant plasma. The detection results of NT-proBNP and BNP were comparable for at least 120 min post sampling and not affected by Hb (2 g/L), DB (428 µmol/L) and chyle (2000 FIU). NT-proBNP was significantly higher in heart failure patients complicated with cerebral infarction (P = 0.003) than in heart failure patients. BNP was significantly higher in heart failure grade III patients complicated with cerebral infarction (P < 0.01). CONCLUSIONS: Cobas E601 and ADVIA Centaur supplied satisfactory detection of NT-proBNP and BNP in patients with chronic heart failure with strong anti-interference capacity. The diagnostic value of NT-proBNP and BNP for chronic heart failure should be analyzed objectively in the presence of complicating diseases.

A novel approach for transferring oleic acid capped iron oxide nanoparticles to water phase.

A novel and simple method was described to transfer oleic acid stabilized iron oxide nanoparticles from organic solutions to water. The oxidation of OA by sodium periodate in mixed solvents formed a carboxyl group or vicinal diol to make the hydrophobic groups to hydrophilic groups on the surface of the nanoparticles. The characterization of nanoparticles indicated that the phase transfer based on the oxidation of OA was successful performed without change in the size and shape of the iron oxide nanoparticles. The hydrophilic groups on the iron oxide surface stabilized the nanoparticles in aqueous solution and the oxidized nanoparticles can be applied to bimolecular immobilization.

Cytotoxicity of Fe3O4/Au composite nanoparticles loaded with doxorubicin combined with magnetic field.

GoldMag (Fe3O4/Au) nanoparticles have the advantages of both magnetic response in an external magnetic field and the immobilization of molecules on their surface in a single step. The cytotoxicities of GoldMag nanoparticles and GoldMag nanoparticles loaded with doxorubicin (Dox-GoldMag) combined with an external magnetic field were tested in vitro on HepG2 malignant tumor cells. The results showed that cell viability remained above 92% when using GoldMag nanoparticles at a concentration as high as 2.0 mg/ml, suggesting the biocompatibility of the nanoparticles. The IC50 (0.731 microg/ml) of the Dox-GoldMag group was higher than that (0.522 microg/ml) of the Dox group (P < 0. 05). However, the Dox-GoldMag group combined with a magnetic field had an obviously increased inhibition rate for the HepG2 cell line and the IC50 was lower than that of the Dox group (0.421 microg/ml). These results indicated that GoldMag nanoparticles loaded with doxorubicin combined with a permanent magnetic field are more cytotoxic and could be a potential targeted drug delivery system.

[Effect of ClpE depletion on the bacterial protein expression profiles of Streptococcus pneumoniae].

OBJECTIVE: To investigate the effect of ClpE on the protein expression profiles of Streptococcus pneumoniae. METHODS: clpE-deficient Streptococcus pneumoniae strain was constructed by long flanking homology-polymerase chain reaction (LFH-PCR) and identified by PCR and sequencing. The total bacterial proteins were analyzed by two-dimensional gel electrophoresis and imaging analysis, and the differentially expressed protein spots were excised by dot-gel digestion with trypsin. Peptide mass fingerprinting (PMF) was obtained by analysis of the fragment length by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS). The PMF was analyzed using software to identify the proteins. RESULTS: The number of matched protein spots of the two gels was 61%. By sequence database searching, 4 out of the 17 differential protein spots were identified, namely hypoxanthine-guanine, pyrrolidone-carboxylate peptidase1, formate-tetrahydrofolate ligase, and bifunctional protein pyrR. CONCLUSION: clpE gene-deficient Streptococcus pneumoniae expresses fewer kinds of proteins at also lower levels than the wild-type bacteria, suggesting that ClpE allows the bacteria to adapt to different host environments by inducing the expression of special proteins.

[Preparation of hen egg yolk immunoglobulin and partial plasma high abundant protein depletion by prepared chicken immunoglobulin coupled on GoldMag carrier].

AIM: To prepare specific egg yolk immunoglobulin (IgY) against HSA and IgG and to make HSA and IgG depletion research in human plasma by binding it to the surface of GoldMag. METHODS: Laying Roman hens immunized with different antigens, such as HSA, IgG and the mixtures of these proteins were used in the experiments. The best condition of isolating the IgY antibody was studied. The titer and purity of the antibody were examined by indirect ELISA and SDS-PAGE respectively. Then the specific IgY was bound to the surface of GoldMag for HSA and IgG depletion research. RESULTS: 60-120 days after immunization, the titer of the antibody in the plasma against different antigens reached 1:15 000-1:25 000; while the titer in the egg reached 1:10 000-1:25 000. The purity was over 98%. Most of HSA and IgG were depleted from human plasma by IgY connected to GoldMag. CONCLUSION: HSA and IgG can be used as antigens to immunize laying hens to produce IgY with high titer and purity. IgY which is bound to the GoldMag carrier can be used to deplete of the relevant HSA and IgG from human plasma effectively. As a new method of studying proteome of plasma, it has some practical applications.

[Cloning the coding cDNA sequence of alpha1, beta2 and gamma2 subunit of GABA-A receptor in American king pigeon].

AIM: To clone and analyse the coding cDNA sequence of alpha1, beta2 and gamma2 subunit of GABAA receptor in American king Pigeon. METHODS: Withdrew total RNA from the American king pigeon brain, reverse transcribing general primers to acquire a gene set cDNA. Designing specific primers of three subunit mRNA of the GABAA receptor, by RT-PCR respectively expanded the conservative gene of al subunit, beta2 subunit and gamma2 subunit of GABAA receptor, and carried on clone, plastid identification and the sequence measurese of three genes. RESULTS: The experiment on sequence measures has succeeded that sequence analysis indicated that lengths of the conservative gene of alpha1 subunit, beta2 subunit and gamma2 subunit of GABAA receptor was respectively 899 bp, 597 bp and 563 bp, homology on reference sequence was respectively 94.99%, 94.64% and 96.28%. CONCLUSION: Homology is high on the conservative gene of alpha1 subunit, beta2 subunit and gamma2 subunit of GABAA receptor of brain tissue of pigeon and chicken but there is a discriminating characteristic in different kinds of animals.

[Preparation of metal chelate affinity chromatographic medium and its application in the purification of 6 x histidine-tagged protein].

Using Sepharose CL-6B as support, 3-Chloro-1, 2-epoxypropane as activated agent, carboxymethylated aspartate (CM-Asp) as chelating ligand, A chelate affinity chromatographic medium based on Co2+, named Co-CM-Asp-Sepharose, was prepared and used to purify 6 x His-tagged fusion proteins. The amount of Co-CM-Asp-Sepharose reacted with 200 microL of lysate, the incubation time, wash condition and the imidazole concentration in the elution buffer were optimized. The purification results using Co-CM-Asp-Sepharose and Ni-NTA-Agarose (product of Qiagen) were compared. The CD155D1 fusion protein was also purified from 5mL of lysate and the amount of protein was determined by Bradford method. The results show that 60 microL of Co-CM-Asp-Sepharose (50% suspension) was suitable for the protein purification from 200 microL of lysate, the optimal incubation time of medium and lysate was 30 min, the optimal imidazole concentration in the eluting buffer was 200 mmol/L, and 200 microg of fusion protein was obtained. In a big scale experiment, 4.6 mg of fusion protein was obtained from 5 mL of lysate using 1.5 mL of Co-CM-Asp-Sepharose (50% suspension). Compared with Ni-NTA-Agarose, the Co-CM-Asp-Sepharose medium exhibits higher selectivity and the protein possesses higher purity.

[Separation and purification of Al13 by chemical precipitation and metathesis].

PACls with different concentrations were prepared by adding sodium carbonate powder into AlCl13 solution. Medium concentration and high Al13 content of PACl was chosen to carry out Al13 separation processes. The influences of SO4/Al molar ratio and the initial total Al concentration on the precipitation reactions of sulfate with different Al species were investigated. The factors influencing the metathesis reaction between solid Al13-SO4 and Ba(NO3)2 were evaluated. Results showed that high Al13 PACl could be obtained at the medium high concentration range of 0.4 - 0.6 mol/L, the optimum SO4/Al ratio was 0.6:1 for precipitation- separation of Al13, Al13 -SO4 precipitates were mostly consisted of tetrahedral crystals. During the metathesis reaction, Ba/SO4 molar ratio of 1:1 is the optimal value. Small range temperature variation and ultrasonic action had no marked influence on metathesis reaction rate and final Al13 concentration. Higher initial Ba(NO3)2 concentration could produce higher concentration Al13 accordingly. The purity of Al13 solution could be reached to 92.1% statistically.

Quantification of prion gene expression in brain and peripheral organs of golden hamster by real-time RT-PCR.

Determination of tissue-specific expression of cellular prion protein (PrPc) is essential for understanding its poorly explained role in organisms. Herein we report on quantification of PrP mRNA in golden hamsters, a popular experimental model for studying mechanisms of transmissible spongiform encephalopathies (TSE), by real-time RT-PCR. Total RNA was isolated from four different regions of the brain and six peripheral organs of eight golden hamsters. PrP mRNA copy numbers were determined using absolute standard curve method with real-time quantitative PCR instrument. It was found that high mRNA levels were present in all four regions of the brain examined, including obex, neocortex, cerebellum, and thalamus. In peripheral organs examined, inguinal lymph node showed high level of the expression similar to that in overall brain; spleen, heart, liver, and lung showed moderate levels of the expression; and kidney showed the lowest expression. Our result is consistent with the potential involvement of different organs in prion diseases and offers essential data for further study of TSE mechanism in this animal model.

Altered expression of the prion gene in rat astrocyte and neuron cultures treated with prion peptide 106-126.

Neuronal degeneration and astrogliosis are hallmarks of prion disease. Synthetic prion protein (PrP) peptide 106-126 (PrP106-126) can induce death of neurons and proliferation of astrocytes in vitro and this neurotoxic effect depends on the expression of cellular PrP (PrPC) and is hence believed to be PrP(C) -mediated. To further elucidate the involvement of PrPC in PrP106-126-induced neurotoxicity, we determined the expression of PrP mRNA in primary culture of rat cortical neuron cells, cerebellar granule cells, and astrocytes following treatment with 50 microM of PrP106-126 scrambled PrP106-126 by quantitative real-time RT-PCR. As shown by MTT test, PrP106-126 induced significant death of neuron cells and marked proliferation of astrocytes after 10 days of treatment. Under the same treatment regimens, the level of PrP gene expression was significantly down-regulated in cortical neuron cell cultures and cerebellar granule cell cultures and was up-regulated in astrocyte cultures. The altered PrP gene expression occurred as early as 3 days after the treatment. After 10 days of treatment, while the cultured cortical neurons underwent further apoptosis, their expression of PrP gene started to recover gradually. These findings indicate that PrP 106-126 regulates transcription of the PrP gene and this activity is associated with its neurotoxicity in primary rat neuronal cultures.