[Esculin inhibits proliferation of triple negative breast cancer cells by down-regulating FBI-1].

Objective: To investigate the effect of esculin on the proliferation of triple negative breast cancer cells and its molecular mechanism. Methods: MDA-MB-231 cells were treated with 28, 56, 112, 225, 450 and 900 µmol/L of esculin for 24, 48 and 72 h, respectively, and the cell viability was detected by cell counting kit 8 (CCK-8) assay. In addition, MDA-MB-231 cells were treated with 0, 225, 450 and 900 µmol/L of esculin for 48 h. And then the changes in cell morphology were observed by inverted microscope. The clone-forming ability was detected by colony formation assay. The mRNA expression levels of FBI-1, p53 and p21 were detected using real-time fluorescence quantitative polymerase chain reaction. The protein expression levels of FBI-1, p53, p21 and Ki67 were detected by western blot. Results: Compared with the blank control group, the cell viability of MDA-MB-231 cells that treated with esculin significantly decreased in a dose-dependent and time-dependent manners. After treatment with esculin, MDA-MB-231 cells shrunk, flattened, adhered poorly to the culture dish and the cell spacing became larger. Meanwhile, shedding and incomplete cells appeared, of which 900 µmol/L of esculin treatment group showed the most dramatic changes. In addition, the colony formation ratios were decreased to (77.18±5.13)%, (65.94±4.98)% and (45.92±3.70)% in the 225, 450 and 900 µmol/L of esculin treatment groups compared with blank control, respectively (P<0.01). Furthermore, the mRNA and protein expressions of FBI-1 increased, while the levels of p53 and p21 mRNA and protein, as well as the protein expression of Ki67 decreased in a concentration-dependent manner (P<0.01). Conclusion: Esculin may regulate cell cycle-related p53-p21 pathway via FBI-1 mediated DNA replication, thus inhibit the proliferation of triple negative breast cancer cells.

[Comparison of modeling effects of two different 7, 12-dimethylbenz[a] anthracene induced breast cancer models in tree shrew].

Objective: To explore the feasibility of 7, 12-dimethylbenz[a] anthracene (DMBA) induced tree shrew breast cancer model, and compare the effects of two administration modes by gavage and mammary gland injection. Methods: A total of 40 tree shrews were randomly divided into two groups (20 animals per group): DMBA gavage group and mammary gland injection group. DMBA was dissolved in edible vegetable oil. For gavage group, tree shrews were administered with DMBA solutions (15 mg/kg) by gavage once a day. For mammary gland injection group, DMBA solution (10 mg/kg) was injected into the mammary fat pad of tree shrews, and the injection was performed for a total of 3 times. From the first administration of DMBA, medroxyprogesterone acetate (MPA, 100 mg/kg) was intramuscularly injected into the muscles of the lateral thighs of tree shrews at the same time, for a total of 5 times. The tumorigenesis and survival of tree shrews were monitored. The tumor histological morphology was observed by HE staining. The expression of estrogen receptor (ER), progesterone receptor (PR), cytokeratin5/6 (CK5/6) and human epidermal factor receptor-2 (HER-2) was detected by immunohistochemical staining. Results: In the gavage group, there were 10 deaths, and 4 tree shrews developed mammary tumors with 20.0% (4/20) tumor formation rate. The success rate of mammary cancer modeling was 10.0% (2/20), and the tumor formation time was 197.3±15.1 days. In the mammary gland injection group, there were 8 tree shrews died, and 9 tree shrews formed tumors with 45.0% (9/20) tumor formation rate. The success rate of mammary cancer modeling was 40.0% (8/20), and the tumor formation time was 71.8±19.0 days. There was no significant difference in mortality and tumor formation rate (P>0.05) between the two groups (all P>0.05). However, in the mammary gland injection group, the success rate of mammary cancer modeling was significantly higher than that in the gavage group (P<0.05), whereas the tumor formation time was markedly shorter than that in the gavage group (P<0.01). The pathological types in the gavage group included ductal hyperplasia, intraductal papilloma and ductal carcinoma in situ, while those in the breast injection group included intraductal papilloma and ductal carcinoma in situ. In both groups, immunohistochemical staining showed the negative expression of HER-2 but positive expression of ER, PR and CK5/6 with varying degrees. Conclusion: Both the DMBA gavage and mammary gland injection can successfully establish the tree shrew breast cancer model, and the modeling effect of mammary gland injection is better than gavage.

[Analyses of the Expression of FBI-1 in Breast Cancer Pre- and Pro-neoadjuvant Chemotherapy].

Objective: To investigate the expression of factor that binds to inducer of short transcripts-1 of HIV (FBI-1)in breast cancer pre- and pro-neoadjuvant chemotherapy and explore the relationship between FBI-1 expression and treatment efficacy. Methods: We collected 50 patients with breast cancer who received neoadjuvant chemotherapy before operation in the Affiliated Tumor Hospital of Guangxi Medical University from January, 2010 to December, 2014. The expression of FBI-1 in breast cancer tissues pre- and pro-neoadjuvant chemotherapy was detected by immunohistochemical staining. We compared the level of FBI-1 expression pre- and pro-neoadjuvant chemotherapy, and tried to explore its relationship with patient and tumor characteristics and treatment efficacy. Results: (1) The rate of upregulated expression of FBI-1 in breast cancer tissues was 70% (35/50). The upregulated expression of FBI-1 was related to the higher clinical stage and trend of lymph node metastasis (P<0.05), whereas not related to the age and expression of ER, PR, Ki-67, and Her-2(P>0.05); (2) the setting of FBI-1 lower expression pre-neoadjuvant chemotherapy had superior treatment outcome than the high expression setting based on either clinical assessment (86.7% vs 51.4%, P=0.027) or pathological assessment(80.0% vs 28.6%, P=0.001); (3) the rate of upregulated FBI-1 expression was significantly decreased post-neoadjuvant chemotherapy(70.0% vs 38.0%, P=0.004), with FBI-1 expression of 22 patients downregulated (62.9%); (4) the expression of FBI-1 in responded setting was significantly decreased than that in the non-responded setting based on either clinical (77.4% vs 26.3%, P=0.001) or pathological (72.7% vs 39.3%, P=0.024) assessment. The downregulation of FBI-1 was correlated to either clinical efficacy (r=0.440, P<0.01) or pathological efficacy (r=0.491, P<0.05) of neoadjuvant chemotherapy. Conclusion: In breast cancer patients receiving neoadjuvant chemotherapy, the upregulated expression of FBI-1 in breast cancer lesion is associated with clinical stage and lymph node metastasis. The neoadjuvant chemotherapy can significantly reduce the expression of FBI-1. The upregulated expression of FBI-1 may be predictive of resistance to chemotherapeutic drugs, and has predictive value for the efficacy of neoadjuvant chemotherapy in breast cancer patients.

[Characteristics and drug resistance of non-O157 Shiga toxin-producing E. coli in animal feces, from Shandong Province].

Objective: To understand the infection status, characteristics and drug resistance of non-O157 Shiga toxin-producing E. coli (STEC) in animal feces in Shandong Province. Methods: From 2015 to 2016, convient sampling method was used to collect 1 022 fresh feces of animals in Weishan county and Laizhou city, and 24 non-O157 STEC were isolated. The serotypes of non-O157 STEC strains were confirmed through serum agglutination test. The susceptibility was explored through the antimicrobial sensitivity experiments. ESBLs activity was confirmed by double-disc diffusion. PCR method was used to detect the resistance genes. PFGE typing was operated to assess the relatedness and variability of the strains. The multi-locus sequence typing (MLST) was adopted to get the allelic profile and ST sequence of strains. Analysis was made on the evolutionary relationship between different ST groups was made through CLC Sequence Viewer and Counting Express. Results: A total of 24 non-O157 STEC were isolated from animal feces. 23 strains were from pig feces, and 1 strain was from cow feces, and the serotypes were more dispersed. All of the 24 strains carried stx2 genes. The highest resistance rate was sulfamethoxazole(22 strains), the mount of cotrimoxazole and nalidixic acid was 18 strains, chloramphenicol was 13 strains, tetracycline was 19, and there was a phenomenon of multiple drug resistance. The drug resistance spectrum was sulfamethoxazole tetracycline-compound novammin-naphthidine-chloramphenicol. All strains were sensitive to cefepime and imipenem. The ESBLs confirmatory test showed that 4 strains of non O157 STEC produced beta lactamase. PCR detected 7 resistance genes, and 4 tetracycline resistance genes (Tet A, Tet B, tetC and tetD) were detected. The beta lactamase resistance genes (blaSHV-1, bla CTX-M, bla TEM) were all negative. 24 strains were divided into 15 PFGE types, and their clustering results were more dispersed and no dominant PFGE type. There were 11 kinds of MLST types, most of them are ST540 and ST5133 types, each of which was 4 strains, and clustered into 1 MLST genomes. Conclusion: The serotypes of non-O157 STEC in animal feces O157 STEC were dispersed, and the resistant rate to common antibiotic was high. MLST typing results presents obvious polymorphism. Surveillance and manage ment of these strains should be strengthened.

The intervention of enalapril maleate and folic acid tablet on the expressions of the GRP78 and CHOP and vascular remodeling in the vascular smooth muscle cells of H-hypertensive rats with homocysteine.

OBJECTIVE: GRP78 and CHOP play essential roles in endoplasmic reticulum stress (ERS) of the vascular smooth muscle cells. We aim to investigate the effect of enalapril maleate and folic acid tablet on the expressions of GRP78 and CHOP and vascular remodeling in a homocysteine (HCY)-treated hypertensive rat model. MATERIALS AND METHODS: The hypertensive rat model was established with the technique of coarctation in the abdominal aorta, and the blood pressure of the rat was measured with the non-destructive tail-cuff method two weeks after operation. Thirty-six rats with hypertension were randomly divided into 3 groups (n=12 in each group). The control group received common diet and double distilled water, methionine group received 30 g/L methionine diet and double distilled water, while enalapril maleate and folic acid tablet group received 30 g/L methionine diet and 0.2 mg.kg-1.d-1 solution of enalapril maleate and folic acid tablet. Samples were collected at week 4 and week 8 for analysis. The plasma homocysteine was measured by homocysteine detector; MAP was detected through carotid artery incubation and aortic media thickness was determined by image analyses software. The expression of GRP78 and CHOP in the vascular smooth muscle cells were identified by immunohistochemistry and Western blot. RESULTS: Compared with the control group, the concentration of HCY in the serum of rats in methionine group was increased significantly after 4 weeks (p < 0.01), and even more significant after 8 weeks (p < 0.01). Compared with that of methionine group, the level of HCY in enalapril maleate and folic acid tablet group rats was significantly decreased (p < 0.01). The level of MAP in methionine group was increased significantly after 8 weeks compared with that of control group (p < 0.05). However, the MAP in enalapril maleate and folic acid tablet group was decreased significantly compared with that of methionine group. Compared with control group, the media thickness of vascular smooth muscle of rats in the methionine group was increased significantly (p < 0.05) while was statistically reduced in the enalapril maleate and folic acid tablet group (p < 0.05). The expressions of GRP78 and CHOP in methionine group were significantly elevated compared to that of control in a time dependent manner (p < 0.05), which were remarkably down regulated in enalapril maleate and folic acid tablet group compared with that in methionine group. CONCLUSIONS: The administration of enalapril maleate and folic acid tablet can maintain the normal state of cells via the alleviation of ERS and vascular damages, reduction of HCY and the thickness of arterial media as well as the improvement of vascular remodeling.

Ta2O5-TiO2 Composite Charge-trapping Dielectric for the Application of the Nonvolatile Memory.

The charge-trapping memory devices with a structure Pt/Al2O3/(Ta2O5) x (TiO2) 1-x /Al2O3/p-Si (x = 0.9, 0.75, 0.5, 0.25) were fabricated by using rf-sputtering and atomic layer deposition techniques. A special band alignment between (Ta2O5) x (TiO2) 1-x and Si substrate was designed to enhance the memory performance by controlling the composition and dielectric constant of the charge-trapping layer and reducing the difference of the potentials at the bottom of the conduction band between (Ta2O5) x (TiO2) 1-x and Si substrate. The memory device with a composite charge storage layer (Ta2O5) 0.5 (TiO2) 0.5 shows a density of trapped charges 3.84 × 1013/cm2 at ± 12 V, a programming/erasing speed of 1 µs at ± 10 V, a 8% degradation of the memory window at ± 10 V after 104 programming/erasing cycles and a 32% losing of trapped charges after ten years. The difference among the activation energies of the trapped electrons in (Ta2O5) x (TiO2) 1-x CTM devices indicates that the retention characteristics are dominated by the difference of energy level for the trap sites in each TTO CTM device.

[The treatment efficiency of a new ear moding device in the infants with congenital ear abnormalities].

Objective:To observe the nonsurgical treatment effciency of a new ear moding device on congenital auricle deformities in order to promote clinical application. Method:Twenty-nine patients (38 ears) from Beijing Tongren Hospital Outpatient received ear molding treatment using the EarWell Infant Ear Correction System. We keep regular follow-up and close observation during the moding period. The treatment effciency was judged by the otologist, plastic surgeons and parents based on the preprocedure and postprocedure photographs and divided into 3 grades: excellent, good and poor. Result:Twenty-nine patients (38 ears) including prominent ear, 2 ears; cup ear,7 ears; lidding/lop ear deformities, 4 ears; Stahl's ear, 4 ears; helical rim abnormalities, 4 ears; conchal crus ear, 3 ears, mixed ear deformities 4 ears; cryptotia, 5 ears; ear malformation, 5 ears, 2 patients (2 ears) stop moding after 3 days treatment due to the low compliance of the infants, the remaining 36 ears received ear molding all have improved. The success rate of the EarWell Infant Ear Correction System is more than 94% (good to excellent). Conclusion:EarWell Infant Ear Correction System have a significant moding effect and can achieve satisfactory results in early time. EarWell system has a high success rate in the treatment of neonatal auricle deformations and mild auricle malformations, depending on the severity of the deformations and the initiation of treatment time. The sooner the noninvasive moding begins (especially within one week after birth), the better effect and the shorter treatment time the patients will achieve.

Expression and clinical significance of Kelch-like epichlorohydrin-associated protein 1 in breast cancer.

Our objective was to explore the expression and clinical significance of Kelch-like epichlorohydrin-associated protein 1 (Keap1) in breast cancer tissue. Eighty-one breast cancer patients having undergone surgical treatment in our hospital between March 2002 and December 2008 were enrolled in this study. Normal tissue adjacent to tumors was used for the control samples. Diagnoses for all patients were confirmed by postoperative pathological examination. Immunohistochemical assays were used to measure the expression of Keap1 protein in breast cancer tissue and adjacent normal tissue, and its clinical significance was explored. We observed that 24.6% breast cancer tissue samples were positive for Keap1, a significantly lower proportion than that seen with adjacent normal tissue specimens (80.2%; P < 0.05). The presence of Keap1 expression did not correlate with age, tumor size, pathological classification, or degree of differentiation. However, it was found to be significantly associated with tumor-node-metastasis stage and the presence of lymphatic metastasis. Kaplan-Meier survival analysis showed a remarkably higher five-year survival rate among patients with positive Keap1 expression than in those lacking detectable levels of the protein (P = 0.032). Keap1 expression is significantly decreased in breast cancer tissue; therefore, the early detection of its expression might have great significance in determining prognosis for breast cancer patients.

[Efficacy observation of (125)I seed implantation therapy for locoregional recurrent and metastatic breast cancer].

OBJECTIVE: To assess the efficacy and side effects of (125)I seed implantation for locoregional recurrent and metastatic breast cancer, and to discuss its role in the comprehensive therapy of breast cancer. METHODS: Forty-three patients with locoregional recurrent or metastatic breast cancer were included in this study. They received (125)I seed implantation and were followed up to evaluate the efficacy and adverse reactions of the treatment. RESULTS: Among 54 lesions in the 43 cases, there were complete response (CR) in 39, partial response (PR) in 13, stable disease (SD) in 2 patients, with a response rate of 96.3%. All 17 cases with local pain achieved pain relief. With a median follow up of 36 months (range 14 to 60 months), the 1-, 3-, and 5-year local control rate was 85.2%, 53.7% and 1.9%, and the 1-, 3-, and 5-year survival rate was 95.3%, 67.4% and 37.2%, respectively. No serious radiotherapy side effect was observed. CONCLUSION: In patients with unresectable locoregional recurrent or metastatic breast cancer, (125)I seed implantation shows proved efficacy and few complications, and can be an important treatment option.

Axillary fine needle aspiration cytology is a sensitive and highly specific technique for the detection of axillary lymph node metastasis: a meta-analysis and systematic review.

BACKGROUND: Fine needle aspiration cytology (FNAC) of axillary lymphadenopathy is a helpful tool in the pre-operative diagnosis of breast cancer patients with axillary lymphadenopathy. To date, no published meta-analysis or systematic review has been performed to assess its overall value. We therefore conducted a meta-analysis to establish the overall diagnostic value of FNAC for axillary lymph node metastasis. METHODS: After a review and quality assessment of 31 studies, published either in Chinese or English, the sensitivity, specificity and other measurements of accuracy of FNAC of axillary lymphadenopathy were pooled using random-effects models. A summary of the receiver-operating characteristic curves was used to summarize overall accuracy. RESULTS: We provided the following estimated values for FNAC in the diagnosis of axillary lymph node metastasis: sensitivity, 0.63 [95% confidence interval (CI), 0.61-0.65]; specificity, 0.99 (95% CI, 0.99-0.99); positive likelihood ratio, 26.52 (95% CI, 18.42-38.18); negative likelihood ratio, 0.34 (95% CI, 0.29-0.40); diagnostic odds ratio, 76.73 (95% CI, 51.98-113.28). CONCLUSIONS: FNAC has adequate sensitivity and high specificity in the diagnosis of axillary lymph node metastasis. A positive axillary FNA result could potentially alter disease management.

Impact of metabolic syndrome components on incident stroke subtypes: a Chinese cohort study.

Limited evidence is available on the risk differences in the development of stroke subtypes in relation to particular clustering patterns of the metabolic syndrome (MetS) components. A follow-up study of a Chinese cohort involving 10,292 individuals was performed to assess the roles of cluster patterns of the MetS components in the prediction of incident stroke subtypes. During follow-up, there were 161 incident cases of ischemic strokes and 41 incident cases of hemorrhagic strokes. Among MetS components, only the hypertensive trait was associated with significantly elevated risks of both ischemic and hemorrhagic strokes. Furthermore, MetS with hypertension as components was associated with increased risk of ischemic and hemorrhagic strokes (adjusted hazards ratio (95% confidence interval) was 2.96 (1.94-4.50) and 2.93 (1.25-6.90), respectively) as compared with those who had neither hypertension nor MetS. Notably, as the number of the MetS components increased, the risk of ischemic stroke significantly and dose-dependently increased. This implies a cumulative effect of MetS components in elevating the risk of ischemic stroke. These findings suggest that MetS comprises heterogenous clusters with respect to the risk of developing the subtype of stroke.

Myostatin gene organization and nodavirus-influenced expression in orange-spotted grouper (Epinephelus coioides).

The relationship(s) between nodavirus infection and myostatin expression in the skeletal muscle tissue of grouper is unclear. To investigate, the grouper (Epinephelus coioides) myostatin gene was cloned and cDNA was utilized to examine the expression of the gene in skeletal muscle and serum of healthy (uninfected) grouper and fish naturally infected with nodavirus. The myostatin gene comprises three exons and two introns and is transcribed as a 2778-bp mRNA length that encodes a 376-aa precursor protein. All exon-intron boundaries conformed to the consensus sequences. Alignment of the upstream sequences indicated that the grouper myostatin promoter has been highly conserved during evolution. Sequence analyses of 1936 bp of the upstream region revealed ten E-box motifs. The protein was consistent with the predicted molecular weight (approximately 42 kDa) of the unprocessed monomeric precursor protein and the processed myostatin form of the protein secreted into the plasma. Transient transfection studies revealed that the activity of the myostatin promoter decreased in a subset of viral titers. Grouper naturally infected with nodavirus displayed downregulation of the myostatin protein.

Factors influencing arsenic accumulation by Pteris vittata: a comparative field study at two sites.

This study compared the factors influencing arsenic (As) accumulation by Pteris vittata at two sites, one containing As along with Au mineralization and the other containing Hg/Tl mineralization. The soils above these two sites contained high As concentrations (26.8-2955 mg kg(-1)). Although the As concentration, pH, soil cation exchange capacity and plant biomass differed significantly between the two sites, no differences were observed in the As concentrations in the fronds and roots, or the translocation factors, of P. vittata, suggesting that this species has consistent As hyperaccumulation properties in the field. The As concentration in the fronds was positively related to phosphorus (P) and potassium (K), but negatively related to calcium (Ca), at one site. This suggested that P, K and Ca influenced As accumulation by P. vittata in the field.

Oxidative stress in human aging and mitochondrial disease-consequences of defective mitochondrial respiration and impaired antioxidant enzyme system.

Respiratory function of mitochondria is compromised in aging human tissues and severely impaired in the patients with mitochondrial disease. A wide spectrum of mitochondrial DNA (mtDNA) mutations has been established to associate with mitochondrial diseases. Some of these mtDNA mutations also occur in various human tissues in an age-dependent manner. These mtDNA mutations cause defects in the respiratory chain due to impairment of the gene expression and structure of respiratory chain polypeptides that are encoded by the mitochondrial genome. Since defective mitochondria generate more reactive oxygen species (ROS) such as O2- and H2O2 via electron leak, we hypothesized that oxidative stress is a contributory factor for aging and mitochondrial disease. This hypothesis has been supported by the findings that oxidative stress and oxidative damage in tissues and culture cells are increased in elderly subjects and patients with mitochondrial diseases. Another line of supporting evidence is our recent finding that the enzyme activities of Cu,Zn-SOD, catalase and glutathione peroxidase (GPx) decrease with age in skin fibroblasts. By contrast, Mn-SOD activity increases up to 65 years of age and then slightly declines thereafter. On the other hand, we observed that the RNA, protein and activity levels of Mn-SOD are increased two- to three-fold in skin fibroblasts of the patients with CPEO syndrome but are dramatically decreased in patients with MELAS or MERRF syndrome. However, the other antioxidant enzymes did not change in the same manner. The imbalance in the expression of these antioxidant enzymes indicates that the production of ROS is in excess of their removal, which in turn may elicit an elevation of oxidative stress in the fibroblasts. Indeed, it was found that intracellular levels of H2O2 and oxidative damage to DNA and lipids in skin fibroblasts from elderly subjects or patients with mitochondrial diseases are significantly increased as compared to those of age-matched controls. Furthermore, Mn-SOD or GPx-1 gene knockout mice were found to display neurological disorders and enhanced oxidative damage similar to those observed in the patients with mitochondrial disease. These observations are reviewed in this article to support that oxidative stress elicited by defective respiratory function and impaired antioxidant enzyme system plays a key role in the pathophysiology of mitochondrial disease and human aging.

Detection and identification of Mycobacterium tuberculosis by nested PCR assays in cerebrospinal fluid samples from patients with suspected tuberculous meningitis.

We used the nested polymerase chain reaction (PCR) assays developed previously to detect and identify Mycobacterium tuberculosis (M. tuberculosis) in the cerebrospinal fluid (CSF) samples from patients with suspected tuberculous meningitis and non-tuberculous patients. Our nested PCR assays target the multi-copy IS6110 insertion element and the single-copy mtp40 genomic DNA of M. tuberculosis. These assays, when used in combination, allowed us to detect a very low number of M. tuberculosis in the CSF samples, which otherwise would be undetectable by the culture method, and to distinguish M. tuberculosis from M. bovis. We applied these nested PCR assays to analyze eleven CSF samples. Among these, five of them were from patients with suspected tuberculous meningitis but all except one were culture negative. Our results of PCR assays show that three of these five are M. tuberculosis positive, one of which is M. bovis positive, and only one is M. tuberculosis negative. The other six CSF samples were from the clinically diagnosed non-tuberculous patients. Surprisingly, two of these so called non-tuberculous patients, a subarachnoid hemorrhage (SAH) and the syndrome of inappropriate antidiuretic hormone secretion (SIADH), were shown M. tuberculosis positive. This finding supports a long-standing argument that tuberculous meningitis is one of the causes of these neurological diseases. These nested PCR assays thus provide the neurologists with an important adjunct, in addition to the patient's clinical presentation and laboratory data, for the diagnosis of tuberculous meningitis.

Comparison of the sensitivity and specificity of an automatic ligase chain reaction assay system with a one-step polymerase chain reaction assay in the diagnosis of Mycobacterium tuberculosis complex.

BACKGROUND: Polymerase chain reaction (PCR) and ligase chain reaction (LCR) are two nucleic acid amplification-based molecular methods. The former has been used widely in the identification of Mycobacterium tuberculosis (M. tuberculosis). In contrast, the LCR assay which was recently introduced is not well known in our medical communities in Taiwan. To determine which method is more reliable and suitable for the identification of M. tuberculosis in our clinics, we compared the sensitivity and specificity of these two methods. METHODS: An automatic LCR assay system and a manual one-step PCR assay were studied in a side by side comparison of their performance in detection of M. tuberculosis. The automatic LCR system uses the single copy antigen protein b (Pab) gene and the manual one-step PCR assay uses the multi-copy IS6110 insertion element as the target DNA; both target DNA sequences are found specifically in M. tuberculosis complex. RESULTS: Both assays detected two of the M. tuberculosis complex strains, M. tuberculosis and M. bovis, but not other mycobacterial strains. In addition, both methods, which were based on different amplification principles, showed compatible sensitivity; as low as 10 and 100 copies of M. tuberculosis genomes were detected by the LCR and PCR assays, respectively. When the template DNA was less than 1000 copies, however, the automatic LCR assay system showed a lower reproducibility than that of the one-step PCR assay. CONCLUSION: Our results suggest that in addition to the PCR assay, the LCR assay is a useful method for the molecular identification of M. tuberculosis complex strains.

Determination of the sensitivity and specificity of PCR assays using different target dnas for the detection of Mycobacterium tuberculosis.

To establish a sensitive, specific and reproducible PCR assay for the detection of Mycobacterium tuberculosis, we evaluated three target DNAs: IS6110, 65 kDa heat shock protein gene; and mtp40 genomic fragment. We purified genomic DNA from 15 mycobacterial strains including four M. tuberculosis isolates, four M. bovis BCG isolates, and one of each for M. fortuitum, M. avium, M. intracellulare, M. szulgai, M. scrofulaceum, M. chelonei, and M. gordonae from the culture and used them as the template DNA. We studied 3 primer sets for IS6110, 2 primer sets for 65 kDa, and 3 primer sets for mtp40. Depending on the assay, these primer sets were used in the single-step PCR and/or nested PCR. The PCR assay targeting the 65 kDa protein gene could detect all of the tested mycobacterial strains, whereas targeting the IS6110 sequence resulted in detection of only M. tuberculosis and M. bovis BCG. Furthermore, targeting the mtp40 genomic fragment could be used to distinguish M. tuberculosis from M. bovis BCG. Using a nested PCR assay with primer sets specifically targeting the IS6110 or 65 kDa, we have been able to detect single copy M. tuberculosis genomic DNA. When the mtp40 genomic fragment was used as the target DNA, the sensitivity of detection was 10 copies of M. tuberculosis genomic DNA. This assay was demonstrated to have good sensitivity and specificity for detection and discrimination of mycobacterial species, and could be used in analyzing the clinical samples with low copy number infections such as the cerebrospinal fluid from the patient with tuberculous meningitis.

Reconstruction of scalp defects with free flaps in 30 cases.

29 consecutive patients treated for reconstruction of various scalp defects with 30 free flaps were reviewed. The scalp defects resulted from accidents (13), electric burns (4), tumour excision (8), chronic osteomyelitis (1), and osteoradionecrosis (1). Secondary reconstructions for cosmetic improvement were performed in 2 patients. The defects involved scalp with bone exposure in 21 patients, and both scalp and calvarium in 8 patients. The average extent of the defects was 130 cm2 (23-420 cm2). Free flaps employed for reconstruction included radial forearm flaps (15), latissimus dorsi muscle flaps (10), medial arm flaps (2), juri flap (1), rectus abdominis muscle flap (1), and scapular flap (1). In 6 cases bone grafts were used for skull reconstruction. Three patients required dura repair. There were two flap failures. Donor-site morbidity was negligible. No local recurrence occurred in 7 tumour cases who are still alive. Secondary procedures (tissue expansion, debulking) were performed in 6 patients. The authors recommend selection of reconstructive options for scalp defects according to their aetiology, localisation, and duration of treatment, whereas the size of the defect dose not seem to be the most important determinant. They conclude that a free flap procedure is appropriate for scalp reconstruction in trauma, osteomylitis, and osteoradionecrosis cases, and following radical resection of malignant tumours.