A 15-year-long Southern blotting analysis of FMR1 to detect female carriers and for prenatal diagnosis of fragile X syndrome in Taiwan.

Here, we review the results of Southern blotting analyses of the FMR1 gene performed in our reference laboratory in Taiwan over a 15-year period. In total, 725 high-risk women with a family history of fragile X syndrome (FXS) or idiopathic intellectual disability, 3911 low-risk pregnant women without such family history, and prenatal diagnosis data for 32 foetuses from 24 carrier mothers were included. Only 2 carriers were in the low-risk group, which indicated a prevalence of 1 of 1955 women (95% confidence interval: 1/7156-1/539). A total of 100 carriers were found to be in the high-risk group, thus revealing a significantly higher frequency than the low-risk group (100/725 vs 2/3911, P<0.0001). Eight of the 14 foetuses that inherited the maternal mutant allele were verified to have a full mutation, with the smallest maternal pre-mutation allele carrying 56 CGG repeats. The overall findings confirmed that the carrier prevalence among low-risk women in Taiwan is significantly lower than that reported in western countries. Therefore, the most important step for preventing FXS in Taiwan would be to focus on high-risk women by promoting general awareness of this disease and spreading knowledge regarding the benefits of carrier screening and prenatal testing.

Microfluidic assisted synthesis of multi-functional polycaprolactone microcapsules: incorporation of CdTe quantum dots, Fe3O4 superparamagnetic nanoparticles and tamoxifen anticancer drugs.

This paper demonstrates a proof-of-concept approach for encapsulating the anticancer drug tamoxifen, Fe3O4 nanoparticles (NPs) and CdTe quantum dots (QDs) into size-controlled polycaprolactone (PCL) microcapsules utilizing microfluidic emulsification, which combined magnetic targeting, fluorescence imaging and drug controlled release properties into one drug delivery system. Cross-linking the composite PCL microcapsules with poly(vinyl alcohol) (PVA) tailored their size, morphology, optical and magnetic properties and drug release behaviors. The flow conditions of the two immiscible solutions were adjusted in order to successfully generate various sizes of polymer droplets. The result showed superparamagnetic and fluorescent properties, and was used as a controlled drug release vehicle. The composite magnetic and fluorescent PCL microcapsules are potential candidates for a smart drug delivery system.

Aberrant co-expression of CD19 and CD56 as surrogate markers of acute myeloid leukemias with t(8;21) in Taiwan.

Aberrant antigen expression in acute myeloid leukemia (AML) has been extensively studied in the West with limited reports from Taiwan. We carried out this retrospective study to characterize the frequency and significance of aberrant antigen expression of AML in Taiwan. Among 111 cases, 58 (52%) showed aberrant antigen expression, most frequently CD7 (27%) and CD56 (23%). Aberrant CD7 expression was observed in all non-AML-M3 subtypes, most frequently in AML-M7 (4/6, 67%); while CD19 expression was only observed in AML-M2 (5/36, 14%). CD56 expression was most common in AML-M5 (4/8, 50%). The relative frequency of CD19 and CD56 expression in AML with t(8;21) was higher than those with other chromosomal abnormalities or normal karyotype (P = 0.011 and 0.005, respectively). In non-M3 AML, aberrant antigen expression was identified in 56/96 (58%) cases, in contrast to 2/15 (13%) AML-M3 cases (P = 0.001). CD7, CD19 and CD56 expression was not correlated with remission rate. We concluded that aberrant immunophenotype was more frequent in non-M3 leukemias in Taiwan. The relative frequency of CD19 and/or CD56 expression in AML with t(8;21) was significantly higher than those without this translocation and co-expression of these two antigens may serve as the surrogate markers for AML with t(8;21).

Production of coloured glass-ceramics from incinerator ash using thermal plasma technology.

Incineration is a major treatment process for municipal solid waste in Taiwan. It is estimated that over 1.5 Mt of incinerator ash are produced annually. This study proposes using thermal plasma technology to treat incinerator ash. Sintered glass-ceramics were produced using quenched vitrified slag with colouring agents added. The experimental results showed that the major crystalline phases developed in the sintered glass-ceramics were gehlenite and wollastonite, but many other secondary phases also appeared depending on the colouring agents added. The physical/mechanical properties, chemical resistance and toxicity characteristic leaching procedure of the coloured glass-ceramics were satisfactory. The glass-ceramic products obtained from incinerator ash treated with thermal plasma technology have great potential for building applications.

Plasma vitrification and re-use of non-combustible fiber reinforced plastic, gill net and waste glass.

Fiber reinforced plastic (FRP) composite material has widespread use in general tank, special chemical tank and body of yacht, etc. The purpose of this study is directed towards the volume reduction of non-combustible FRP by thermal plasma and recycling of vitrified slag with specific procedures. In this study, we have employed three main wastes such as, FRP, gill net and waste glass. The thermal molten process was applied to treat vitrified slag at high temperatures whereas in the post-heat treatment vitrified slags were mixed with specific additive and ground into powder form and then heat treated at high temperatures. With a two-stage heat treatment, the treated sample was generated into four crystalline phases, cristobalite, albite, anorthite and wollastonite. Fine and relatively high dense structures with desirable properties were obtained for samples treated by the two-stage heating treatment. Good physical and mechanical properties were achieved after heat treatment, and this study reveals that our results could be comparable with the commercial products.

Sentinel node detection with radiocolloid lymphatic mapping in early invasive cervical cancer.

We assessed the feasibility of sentinel lymph node detection using technicium-99 radiocolloid lymphatic mapping for predicting lymph node metastases in early invasive cervical cancer. Thirty patients with cervical cancer (stages IA2-IIA) underwent preoperative lymphoscintigraphy using technicium-99 intracervical injection and intraoperative lymphatic mapping with a handheld gamma probe. After dissection of the sentinel nodes, the standard procedure of pelvic lymph node dissection and radical hysterectomy was performed as usual. The sentinel node detection rate was 100% (30/30). There were seven (23.3%) cases of microscopic lymph node metastases on pathologic analysis. All of them had sentinel node involvement. Therefore, the sensitivity of sentinel node identification for prediction of lymph node metastases was 100%, and no false negative was found. Preoperative lymphoscintigraphy, coupled with intraoperative lymphatic mapping, located the sentinel nodes accurately in our study patients. This sentinel node detection method appears to be feasible for predicting lymph node metastases.

Determination of urinary beryllium, arsenic, and selenium in steel production workers.

Some of the most pernicious dangers of pollution arise from the presence of traces of toxic elements in the environment. In this work, we report on the determination of beryllium, arsenic, and selenium in the urine of steel production and steel quality control (QC) workers, in comparison to healthy control subjects. The urine samples were digested by a microwave system. Graphite furnace and hydride atomic absorption was used for the quantitative measurements of Be and As and Se, respectively. A quality control method for these procedures was established with concurrent analysis of Standard Trace Metals 7879 Level II and NIST SRM 2670 (Toxic Elements in Freeze Dried Urine). The results show that the urinary levels of these elements in steel production (As, 38.1 +/- 28.7 microg/L; Be, 1.58 +/- 0.46 microg/L, and Se, 69.2 +/- 28.8 +/- g/L) and in quality control workers (As, 23.9 +/- 18.1 microg/L; Be, 1.58 +/- 0.46 microg/L, and Se, 54.8 +/- 25.1 microg/L) are significantly higher than in the controls (As, 10.3 +/- 8.7 microg/L; Be, 0.83 +/- 0.46 microg/L; and Se, 32.3 +/- 13.5 microg/L). The possible connection of these elements with the etiology of disease and the possible role of selenium as a protective agent against the oncogenic and teratogenic action of other substances is discussed. We suggest the need for improvement of environmental conditions in the workplace through better ventilation and industrial hygiene practices.

Treatment and recycling of incinerated ash using thermal plasma technology.

To treat incinerated ash is an important issue in Taiwan. Incinerated ashes contain a considerable amount of hazardous materials such as dioxins and heavy metals. If these hazardous materials are improperly treated or disposed of, they shall cause detrimental secondary contamination. Thermal plasma vitrification is a robust technology to treat and recycle the ash residues. Under the high temperature plasma environment, incinerated ashes are vitrified into benign slag with large volume reduction and extreme detoxification. Several one-step heat treatment processes are carried out at four temperatures (i.e. 850, 950, 1,050 and 1,150 degrees C) to obtain various "microstructure materials". The major phase to form these materials is a solid solution of gehlenite (Ca2Al2SiO7) and åkermanite (Ca2MgSi2O7) belonging to the melilite group. The physical and mechanical properties of the microstructure materials are improved by using one-step post-heat treatment process after plasma vitrification. These microstructure materials with good quality have great potential to serve as a viable alternative for construction applications.

Ovarian fibrosarcoma with long-term survival: a case report.

Primary ovarian fibrosarcoma is an exceedingly rare malignant ovarian stromal tumor which has a poor prognosis. We report here a 46-year-old woman who suffered from irregular vaginal bleeding for 2 months. She received hysterectomy and salpingo-oophorectomy due to a provisional diagnosis of uterine and ovarian tumors. At surgery, an 8-cm ovarian solid multilobular tumor was found. Frozen section examination revealed an ovarian fibrosarcoma. She then underwent staging procedures including intraperitoneal washing, cytology, and pelvic and para-aortic lymph node sampling. Final pathologic examination revealed that the tumor exhibited densely packed spindle cells in storiform configuration with obvious increased mitotic activity. In addition, the flow cytometric study showed marked elevated percentage of tumor cells in the S phase (13.1%). After surgery, the patient received six courses of combination chemotherapy with epirubicin, ifosfamide, and dacarbazine (DTIC). The patient stood the treatment well and is free from disease 6 years later.

Synthesis and antibacterial evaluation of certain quinolone derivatives.

A number of 7-substituted quinolone derivatives were synthesized and evaluated for antibacterial and cytotoxic activities. Preliminary results indicated that most compounds tested in this study demonstrated better activity against methicillin-resistant Staphylococcus aureus than norfloxacin. Among them, 1-(4-amino-2-fluorophenyl)-6-fluoro-1,4-dihydro-7-[4-[2-(4-methoxyphenyl)-2-hydroxyiminoethyl]-1-piperazinyl]-4-oxo-3-quinolinecarboxylic acid (11d) and its ketone precursor 10d exhibited significant activities against Klebsiella pneumoniae, methicillin-resistant S. aureus, erythromycin- and ampicillin-resistant Streptococcus pneumoniae, and vancomycin-resistant Enterococcus faecalis. Due to strong cytotoxicities of 11d (a mean log GI(50) of -5.40), compound 10d, with good antibacterial activities and low cytotoxicities (a mean log GI(50) of -4.67), is a more potential drug candidate.

Plastic microchip electrophoresis for genetic screening: the analysis of polymerase chain reactions products of fragile X (CGG)n alleles.

Clinical screening of abnormal chromosomes associated with fragile X syndrome (FXS) demands a high-throughput method including DNA sizing and detection of the amplified products. This study is to explore the use of polymer microchip electrophoresis for the analysis of polymerase chain reaction (PCR) products of fragile X (CGG)n alleles to facilitate a fast exclusion test of FXS. The sequences flanking the CGG-repeat of FMR1 gene was amplified by betaine-PCR and the amplified products were desalted and then analyzed by microchips which were fabricated on poly(methyl methacrylate) (PMMA) substrate. The PCR bands with more than six CGG-repeats in difference could be clearly distinguished in less than 3 min by microchip electrophoresis with a separation length of 6 cm. It was found that the signal was greatly enhanced with the use of both covalent (Cy5) and intercalating dye (TORRO-3), which has never been demonstrated before. We tested the method by reanalysis of twelve samples from males and six samples from females. For female samples with less than six repeat differences, Southern blotting method was performed to confirm or exclude the findings from microchips. It was found that the test results from all male and female samples show a 100% correlation between the microchip electrophoresis and the existing methods.

Inhibition of the expression of inducible nitric oxide synthase and cyclooxygenase-2 in macrophages by 7HQ derivatives: involvement of IkappaB-alpha stabilization.

Nitric oxide is an important biological mediator associated with multiple pathophysiological phenomena, such as platelet aggregation, vasodilation, septic shock, and autoimmune diseases. Prostaglandins, derived from cyclooxygenases, play prominent roles in homeostasis and inflammation. In this study, we characterized the effects of 7HQ derivatives (7-[(4-methylene-5-oxo-2-R-2-tetrahydrofuranyl) methoxy]-3,4-dihydrocarbostyril, where R is methyl, phenyl, p-fluorophenyl and p-phenylphenyl; 7HQ-1,-2,-3 and-4, respectively) in murine RAW 264.7 cells, a macrophage-like cell line. Lipopolysaccharide, the active component of endotoxin, significantly induced the expression of inducible nitric oxide synthase and cyclooxygenase-2, leading to the accumulation of nitrite and prostaglandin E(2), respectively. These actions of lipopolysaccharide were inhibited by 7HQ derivatives; additionally, the inhibition of the expression, rather than the activity, of inducible nitric oxide synthase correlated well with that of nitric oxide formation. Western blotting and electrophoretic mobility shift assay results demonstrated that the 7HQ derivatives could effectively inhibit IkappaB-alpha degradation and nuclear factor kappaB (NF-kappaB) translocation. At higher concentrations, 7HQ derivatives also inhibited cyclooxygenase-2 enzyme activity. These results suggest that 7HQ derivatives exhibit inhibitory effects on lipopolysaccharide-induced nitric oxide production and expression of inducible nitric oxide synthase and cyclooxygenase-2 through inhibition of IkappaB-alpha degradation and NF-kappaB activation.

An effective strategy of using molecular testing to screen mentally retarded individuals for fragile X syndrome.

Fragile X syndrome (FXS) is the most common form of familial mental retardation (MR). It is caused by the expansion of the CGG repeat in the FMR1 gene on the X chromosome. To date, FXS is not treatable, but can be prevented by prenatal genetic examination. Identifying women who carry a full mutation or premutation FMR1 gene is thus very important, and can be done by tracing family members of FXS subjects. However, most of the FXS subjects in Taiwan as well as those in many other countries have not been identified. In this study the authors attempt to develop reliable and inexpensive tests suitable for a large-scale screen of subjects with MR for FXS. Together with their previous study, a total of 311 male and 160 female subjects with MR were screened with nonradioactive Southern blot assay using mixed deoxyribonucleic acid from three subjects of the same sex. From these subjects, nine male subjects and one female FXS subject were diagnosed. All male subjects were also screened with nonradioactive polymerase chain reaction (PCR). These nine male FXS subjects were also detected on the basis of PCR amplification failure. No false-negative results were discerned. The PCR procedure was simplified further by combining it with an analysis of a blood spot on filter paper, which is a much simpler and cheaper method for sample collection and DNA preparation. This method was then used to screen 104 boys with MR. Two of them were suspected, and later confirmed with Southern blot assay, as subjects with FXS. This study suggests that simple PCR combined with blood spot analysis could be a reliable, inexpensive test that is feasible for a large-scale screening of male subjects with MR for FXS. However, Southern blot assay with mixed deoxyribonucleic acid is appropriate for screening female subjects. Based on this strategy, most FXS subjects could be identified easily for further management.

Synthesis, antibacterial, and cytotoxic evaluation of certain 7-substituted norfloxacin derivatives.

We report herein the synthesis and biological evaluation of two series of 7-substituted norfloxacin derivatives. Most compounds tested in this study demonstrated better activity against methicillin-resistant Staphylococcus aureus than norfloxacin. Preliminary in vitro evaluation indicated that the 7-[4-(2-hydroxyiminoethyl)piperazin-1-yl] derivatives 3b-e possess distinct cytotoxicity profiles as compared with their alpha-methylene-gamma-butyrolactone counterparts, 4b,e: i.e., excellent activities against the renal cancer subpanel. Among them, 1-ethyl-6-fluoro-7-¿4-[2-(4-chlorophenyl)-2-hydroxyiminoethyl]-1-p ipe razinyl¿-4-oxo-1,4-dihydro-3-quinolinecarboxylic acid (3d) demonstrated the most significant activities against renal cancer cell lines, with log GI(50) values of -6.40 against CAK-1, -6.14 against RXF 393, and -7.54 against UO-31, compared with a mean log GI(50) value of -5.03.

Synthesis and cytotoxic evaluation of a series of gamma-substituted gamma-aryloxymethyl-alpha-methylene-gamma-butyrolactones against cancer cells.

PURPOSE: The main objective of this investigation was to explore the cytotoxic structure-activity relationships of gamma-substituted gamma-aryloxymethyl-alpha-methylene-gamma-butyrolactones against cancer cells. METHODS: The target compounds were synthesized in two steps commencing with aryl-OH which was treated with a bromomethyl ketone followed by the Reformatsky-type condensation. RESULTS: Seven types of alpha-methylene-gamma-butyrolactones were evaluated in vitro against 60 human cancer cell lines derived from nine cancer cell types. The average values of log GI50 indicated that for the aryl portion, potencies of these alpha-methylene-gamma-butyrolactones are in a decreasing order of quinolin-2(1H)-one (or 2-hydroxyquinoline, 21, -5.89) > quinoline (19, -5.79) > 2-methylquinoline (20, -5.69) >8-hydroxyquinoline (17,-5.64) > 2-naphthalene (16, -5.59) > benzene (15, -4.90). The same order was obtained for both log TGI and log LC50. However, for the gamma-substituent, the potencies are in a decreasing order of biphenyl (16f-21f) > phenyl and 4-substituted phenyl (16b-e-21b-e) > methyl (16a-21a). CONCLUSIONS: Unlike cardiovascular activities of alpha-methylene-gamma-butyrolactones in which a gamma-methyl substituent is necessary for vasorelaxing effect while a phenyl or a halogen-substituted phenyl is prefer for the antiplatelet activities, a gamma-biphenyl substituent proved to be the best for their cytotoxicities against various cancer cell lines tested.

Implication of screening for FMR1 and FMR2 gene mutation in individuals with nonspecific mental retardation in Taiwan.

Fragile X syndrome (FXS) is the most common form of familial mental retardation (MR), attributable to (CGG)n expansion in the FMR1 gene. FRAXE is less frequent, associated with a similar mutation of the FMR2 gene. This study attempted to ascertain the prevalence of both disorders in Taiwan, as well as to develop a method to effectively find carriers. A total of 321 patients with nonspecific MR were screened for the FMR1 and FMR2 mutation. Four of 206 boys and men (1.9%) and 1 in 115 girls and women (0.9%) were identified as having FXS. All four FXS boys or men could be identified by Southern blot analysis, as well as by a simple nonradioactive polymerase chain reaction analysis. None of the 206 boys or men had FMR2 full mutation. This confirmed the low incidence of FRAXE in Chinese. FXS appears to be more prevalent among patients with mild MR, because 4 of the 5 patients with FXS were from the 115 with mild MR (3.48%) and only 1 was from the other 206 with severe MR (0.49%). All five FXS cases were maternally inherited. Other family members were resistant to further searching for carriers. It is worth noting that none of these mothers had a discernible premarital family history of MR. Thus the negative family history could not preclude the possibility that a woman was a carrier. To identify female carriers of childbearing age, beyond the scope of family history, is thus worthy of further exploration. Screening men for carriers using this inexpensive method is probably feasible, even though normal transmitting men have no immediate risk of producing a child with the disease. Female carriers can then be effectively identified from these normal transmitting men and can take all preventive measures.

An efficient one-pot method for the large-quantitative production of radiopharmaceutical ligand: diazadioximes.

An efficient one-pot procedure for the preparation of diazadioxime was described. Treatment of ketooximes with alkyldiamine followed by NaBH4 in dry ethanol afforded the corresponding d,l-diazadioximes in 56--74% yield without isolation of the intermediates.

Cloning of two new cry genes from Bacillus thuringiensis subsp. wuhanensis strain.

With PCR products as probes, we have cloned two new cry-type genes from Bacillus thuringiensis subsp. wuhanensis. The deduced amino acid sequence of the first clone is 77.3% identical to Cry1Ga1. The deduced protein sequence of the second clone is 69.8-78.7% identical to that of Cry1B group. The nomenclature assignment of these two clones is, therefore, named Cry1Gb1 and Cry1Bd1, respectively. The Cry1Bd1 is toxic to Plutella xylostella larvae, and the Cry1Gb1 is toxic to Pieris rapae larvae.

Pilot fragile X screening in normal population of Taiwan.

Fragile X syndrome (FXS) is the most common hereditary form of mental retardation. Molecular analysis of the FMR1 gene has now been applied to diagnosis and carrier detection. Because treatment is not feasible, prevention of FXS by prenatal diagnosis of carrier women early during pregnancy is important. The aim of this pilot study was to ascertain the prevalence of mutant FMR1 gene in normal population of Taiwan and to evaluate the efficacy of a betaine-based polymerase chain reaction (PCR) and nonradioactive Southern blot assays. The DNA was randomly and anonymously collected from 100 women and 100 men. The results showed 62% of the women were heterozygous for the CGG-repeat size in FMR1 gene. One of 300 X chromosomes in this study showed premutation, with 95 CGG repeats. All other chromosomes have CGG repeats ranging from 19 to 52, with eight chromosomes (3%) having more than 40 CGG repeats. The most prevalent allele has 29 repeats (48.1%), followed by 30 (24.0%) and 36 (9.5%), respectively. The results of this study reconfirmed previous reports that the prevalent FMR1 CGG repeat alleles in Chinese population are different from that of other populations. However, the prevalence of premutation gene seems to be comparable among them. The betaine-based PCR could minimize the intrinsic problem of preferential amplification and may reliably determine the different allele repeats in heterozygous females. This nonradioactive Southern blot protocol is safe, efficient, and inexpensive. However, further technical improvement may be needed to be more cost-effective for a wide screening of all pregnant women.