Fluorescence-based Polymerase Chain Reaction Research Articles

P53 loss of heterozygosity (LOH) in formalin-fixed paraffin-embedded leiomyosarcoma (LMS): a novel report.

The occurrence of p53 loss of heterozygosity (LOH) is a common genetic event in malignancy. LOH occurs when a heterozygous locus loses one of its two parental alleles, becoming homozygous at that locus, by either copy number loss (CNL-LOH) or by becoming copy number neutral (CNN-LOH). A role for CNL-LOH (cnLOH) has been postulated in cancer aetiology. Loss of heterozygosity (LOH) results in irreversible genetic loss. LOH was determined in DNA extracted from formalin-fixed paraffin-embedded (FFPE) leiomyosarcoma (LMS) specimens in a retrospective study from 30 patients, to assess the prognostic significance of LOH. The findings were analysed and their validity assessed. LOH was an adverse prognostic factor in LMS. Prospective uniform standardisation of formalin-fixation techniques is required. DNA was extracted from 169 formalin-fixed paraffin blocks of 30 patients with LMS, following extensive tissue microdissection. Genomic DNA was amplified using the polymerase chain reaction (PCR) technique. Fluorescence-based microsatellite PCR was used to detect and quantitate heterozygosity loss. LOH was detected at gene locus 17p13 in 16 LMS (Four grade 2 and 12 grade 3 LMS). LOH was not detected in 14 LMS cases (one grade 1, five grade 2 and eight grade 3 LMS). LOH was associated with shorter patient survival. The results reported herein endorse the value of utilizing FFPE DNA in identifying LOH as a prognostic factor in LMS. The results have implications for tumour biobanking and precision medicine in patients with sarcomas.

Association of two variable number of tandem repeats in the monoamine oxidase A gene promoter with suicide completion: The present study and meta-analysis.

One potential cause of suicide is serotonergic dysfunction. Sex differences have been reported to modulate the effects of serotonergic polymorphisms. Monoamine oxidase A (MAOA) is an enzyme that degrades serotonin and is located on the X chromosome. A previous study indicated that the upstream (u) variable number of tandem repeat (VNTR) in the MAOA gene promoter may be associated with suicide. However, a meta-analysis showed that this polymorphism may not be related to suicide. According to a recent study, compared with the uVNTR, the distal (d)VNTR and the haplotypes of the two VNTRs modulate MAOA expression. We examined the two VNTRs in the MAOA gene promoter in 1007 subjects who committed suicide and 844 healthy controls. We analyzed the two VNTRs using fluorescence-based polymerase chain reaction assays. We conducted a meta-analysis for the two VNTRs to update it. Our results demonstrated that neither the genotype-based associations nor allele/haplotype frequencies of the two VNTRs were significantly associated with suicide. In the meta-analysis, we did not indicate relationships between uVNTR and suicide nor did we identify articles analyzing dVNTR in suicide. Overall, we did not find a relationship between the two VNTRs in the MAOA promoter and suicide completion; thus, warranting further studies are required.

Methylation profiles of global LINE-1 DNA and the GSTP1 promoter region in children exposed to lead (Pb)

ABSTRACT Lead (Pb) exposure has adverse health effects and altered DNA methylation may contribute to Pb toxicity. LINE-1 is an interspersed repeated DNA that is used as a surrogate marker for estimating genomic DNA methylation levels, and GSTP1 is an isozyme that detoxifies xenobiotics like Pb, and its expression is inhibited by methylation. Thus, to assess the effects of Pb exposure on global hypomethylation and gene-specific promoter hypermethylation, we examined DNA methylation at LINE-1 repetitive elements and the GSTP1 promoter region. Blood samples were obtained from children (N = 123) living in Pb-polluted areas (as exposed children) and children (N = 63) living in Pb-unpolluted areas (as control children) in Kabwe, Zambia. ICP-MS was used to determine blood lead levels (BLLs), and pyrosequencing and a fluorescence-based polymerase chain reaction assay were used to determine levels of LINE-1 methylation and GSTP1 promoter methylation, respectively. Inverse association was found between BLLs and LINE-1 methylation (β = – 0.046, p = 0.006). The highest quartile of BLL had significant hypomethylation of LINE-1 (p for trend = 0.03), suggesting the higher the BLL, the lower LINE-1 methylation. GSTP1 methylation levels did not differ significantly between the two areas (p = 0.504), nor was it associated with Pb poisoning risk (OR = 1.03, p = 0.952), indicating GSTP1 methylation may not be a reliable biomarker of Pb exposure in healthy people. Therefore, Pb-related health problems could result from global DNA methylation changes due to high BLLs.

Ligation-Enabled Fluorescence-Coding PCR for High-Dimensional Fluorescence-Based Nucleic Acid Detection.

Polymerase chain reaction (PCR) is by far the most commonly used method of nucleic acid amplification and has likewise been employed for a plethora of diagnostic purposes. Nonetheless, multiplexed PCR-based detection schemes have hitherto been largely limited by technical challenges associated with nonspecific interactions and other limitations inherent to traditional fluorescence-based assays. Here, we describe a novel strategy for multiplexed PCR-based analysis called Ligation-eNabled fluorescence-Coding PCR (LiNC PCR) that exponentially enhances the multiplexing capability of standard fluorescence-based PCR assays. The technique relies upon a simple, preliminary ligation reaction in which target DNA sequences are converted to PCR template molecules with distinct endpoint fluorescence signatures. Universal TaqMan probes are used to create target-specific multicolor fluorescence signals that can be readily decoded to identify amplified targets of interest. We demonstrate the LiNC PCR technique by implementing a two-color-based assay for detection of 10 ovarian cancer epigenetic biomarkers at analytical sensitivities as low as 60 template molecules with no detectable target cross-talk. Overall, LiNC PCR provides a simple and inexpensive method for achieving high-dimensional multiplexing that can be implemented in manifold molecular diagnostic applications.

PCR-coupled Paper-based Surface-enhanced Raman Scattering (SERS) Sensor for Rapid and Sensitive Detection of Respiratory Bacterial DNA

A paper-based surface-enhanced Raman scattering (SERS) substrate composed of silver-nanowires (AgNWs) was coupled with polymerase chain reaction (PCR) for rapid and sensitive determination of respiratory bacterial DNA. EvaGreen dye, which is a DNA intercalating molecule, was introduced to the low-cycled PCR product, and the difference in dye intercalation in the DNA structure was quantified by Raman spectroscopy to verify the presence of a target gene. Mycoplasma pneumoniae (M. Pne), was selected as a target for the sensing demonstration. At various gene concentrations and amplification cycles, detection capability of fluorescence-based real-time PCR (RT-PCR) and the PCR-coupled SERS method were compared to develop a DNA detection system capable of low-cycle amplification and sensitive DNA sensing. After 10 cycles, the PCR-coupled SERS method showed enhanced detection capability with a DNA detection limit of 3.12 pg/μL. Then, the SERS substrate was prepared as a rapid kit that included a test line and negative and positive control lines. Target DNA after 10 cycles of amplification was successfully discriminated compared to non-target DNA and it was statistically relevant. The developed system is expected to be used as a detection method for various bacteria and viruses, and also be integrated with diverse POC diagnostic systems.

Diagnosis and treatment strategy of coronavirus disease 2019 with cardiovascular disease in elderly patients.

Diagnosis and treatment strategy of coronavirus disease 2019 with cardiovascular disease in elderly patients.

Development of High Quality EST-SSR Markers Without Stutter Bands in Peach and Their Application in Cultivar Discrimination and Hybrid Authentication

Peach (Prunus persica) is an important fruit crop worldwide with several thousand cultivars. Cultivar discrimination and hybrid authentication are often required in peach breeding and can be achieved by applying various molecular markers including simple sequence repeat (SSR). In this study a total of 2146 expressed sequence tag (EST)–SSR loci were detected with the 10,737 EST sequences retrieved from the NCBI. A total of 49 EST-SSR markers, including 24 simple ones with a motif comprising of tri-, tetra-, penta-, hexanucleotides, and 25 compound ones, were selected and then primers were designed. Following conventional polymerase chain reaction (PCR) specificity control and sequence authentication, as well as fluorescence-based PCR product size and stutter band evaluation, 37 EST-SSR markers with correct amplification and without stutter band interference were validated. Among them, 14 were polymorphic in 18 closely related peach accessions, with polymorphism information content (PIC) ranging from 0.0994 to 0.3750. The 18 peach accessions can be distinguished using nine polymorphic markers, with the exception of ‘Shangshandayulu’ and ‘Xipu 1’, both being bud sports from ‘Yulu’. The clustering of the accessions as well as the fingerprint profiles supported the authentication of the hybrids. These EST-SSR markers are useful for peach breeding research.

Voice Alterations in Patients With Spinocerebellar Ataxia Type 7 (SCA7): Clinical-Genetic Correlations

Spinocerebellar ataxia type 7 (SCA7) is an inherited neurodegenerative disease caused by the expansion of a cytosine-adenine-guanine triplet located in the coding region of the ATXN7 gene, which is characterized by cerebellar ataxia, pigmentary macular degeneration, and dysarthria. Although dysarthria is a common feature in various SCA, its clinical characterization has been barely approached. In this study, we report, to our knowledge for the first time, a detailed voice analysis in a large series of patients with SCA7, using different vocal parameters, including jitter, shimmer, and fundamental frequency. Patients were molecularly diagnosed using fluorescent-based polymerase chain reaction and capillary electrophoresis, and clinically characterized using the Scale for the Assessment and Rating of Ataxia and the Inventory of Non-Ataxia Symptoms. We found altered jitter, shimmer, and fundamental frequency measurements in patients with SCA7 compared with control subjects (P < 0.05). However, voice impairment was found unrelated with both age at disease onset and size of the cytosine-adenine-guanine triplet tract. Remarkably, jitter and shimmer measurements of patients were found to correlate with their Inventory of Non-Ataxia Symptoms, but not with their Scale for the Assessment and Rating of Ataxia scores, implying that voice impairment is the result of extra-cerebellar manifestations of the disease. We propose that deficiency of the extra-cerebellar component of SCA7 might lead to sudden changes in laryngeal muscle tone, producing instability in sustained vowel phonation. Clinical characterization of voice will help to discriminate SCA7 from other SCA and to guide vocal therapy treatments.

Roles of human papillomavirus infection and stathmin in the pathogenesis of sinonasal inverted papilloma.

The purpose of this study was to investigate roles of human papillomavirus (HPV) infection and stathmin in sinonasal inverted papilloma (SNIP). HPV DNA detection was performed by the fluorescence-based polymerase chain reaction (PCR) method. Stathmin protein expression was investigated by the immunohistochemistry method and mRNA expression of stathmin, Kif2a, and cyclin D1 were assessed by real-time PCR in SNIP and control subjects. The positive rate of HPV DNA detected in SNIP was about 53.6% (15 of 28). Recurrent cases showed a higher rate of HPV infection compared with initial cases and higher Krouse stage (T3 + T4) cases showed higher rate of HPV infection than lower Krouse stage (T1 + T2) cases. Stronger expression of stathmin, Kif2a, and cyclin D1 were observed in SNIP, especially HPV(+) SNIP. HPV infection was closely associated with recurrence and progression of SNIP. Stathmin is a valuable prognostic marker and could be considered as a therapeutic target in patients with SNIP.

Electrochemical method for monitoring the progress of polymerase chain reactions using Methylene blue as an indicator

We report on the proof-of-principle of an amperometric method to monitor the progress of a polymerase chain reaction (PCR) in real-time. It is based on the finding that the intercalating redox probe Methylene Blue (MB) becomes less easily electrochemically detectable once intercalated into double-stranded DNA (ds-DNA) compared to its free form. This was studied by cyclic voltammetry before and after addition of salmon sperm ds-DNA. Under optimized conditions, the products of the PCR of mitochondrial DNA fragments were quantitatively detected at different stages of amplification cycles. This strategy is potentially cheaper and easier to integrate into a hand-held miniaturized device than fluorescence-based real-time PCR.

High-frequency microsatellite instability and BRAF mutation (V600E) in unselected Serbian patients with colorectal cancer

Microsatellite instability (MSI) is a genetic consequence of a MisMatch Repair defect in colorectal cancer (CRC). We compared clinicopathohistological features with MSI status of CRC and evaluated prognostic significance of MSI status and BRAF mutation in the group of MSI-H tumors. 155 primary CRCs were excised surgically, 2006-2008. MSI analysis was carried out using a fluorescence-based pentaplex polymerase chain reaction technique. BRAF mutation (V600E) was analyzed by direct sequencing in MSI-H tumors. For all patients were evaluated: age, gender, localization, tumor cell type, tumor differentiation, mucin production, lymphocytic infiltration (TILs) and TNM stage. Patients' disease-free survival (DFS) was compared according to MSI and BRAF status using Kaplan-Meier test. Of the 155 CRCs, 19 (12.3%) were MSI-H, and 136 (87.7%) were MSS/L. BRAF mutations were found in 4 of the MSI-H tumors. Patients with MSI-H CRC had lower recurrence rate (log rank test; P=0.04) than MSS/L group. Patients with MSI-H tumor and BRAF mutation had worse DFS than MSI-H tumors without this mutation (log rank test; P=0.01). Most of the clinicopathologic characteristics of MSI-H CRC in Serbian patients are similar to those reported in previous studies. Patients with MSI tumor phenotype had favourable prognosis, but in those with BRAF mutation higher recurrence rate was observed.

Impact of DNMT3A mutations on Clinical Response to the Hypomethylating Agent Decitabine in Older Patients (pts) with Acute Myeloid Leukemia (AML)

Abstract 944Loss of function mutations of the DNA methyltransferase 3A (DNMT3A) gene have been associated with a negative impact on outcome in AML pts treated with conventional chemotherapy and/or stem cell transplantation. DNA methyltransferases (DNMTs) such as DNMT3A are responsible for catalyzing the addition of methyl groups to CpG dinucleotides. In AML, aberrant DNMT activity plays a role in epigenetic silencing of genes involved in hematopoiesis, and inhibition of DNMT activity by hypomethylating agents such as azacitidine and decitabine has been explored as a way to reverse hypermethylated gene silencing and induce clinical response. However, whether DNMT3A mutations influence response to hypomethylating agents is unknown. We studied the frequency and impact of DNMT3A mutations in a cohort of AML pts similarly treated with low dose decitabine. Genomic DNA from pretreatment bone marrow specimens from 46 pts (age 32–85) with untreated or relapsed AML who received decitabine 20mg/m2/d for 10 days every 28 days alone (n=39) or in combination with escalating doses of bortezomib (1.3mg/m2/d on days 5, 8, 12 & 15; n=7) were evaluated. DNMT3A was studied for mutations in the “hot spot” regions, and the mutational status of NPM1, CEBPA, IDH1/IDH2 and TET2 was assessed using polymerase chain reaction (PCR) and direct sequencing. Mutations within FLT3 were determined by quantitative fluorescence-based PCR capillary electrophoresis. Of the 46 pts, 17 had cytogenetically normal (CN) AML. Ten pts belonged to the Favorable genetic group as defined by the European LeukemiaNet (ELN) reporting system, nine pts fell into the ELN Intermediate-I, 11 were in the Intermediate-II, and 16 in the Adverse. Of the 46 pts, eight (17%) carried a DNMT3A mutation: six had codon R882 missense mutations, one nonsense (c.1729A>T; p.K576X) and one splice-site (c.2322+1G>A) mutation. DNMT3A mutations were distributed across all ELN genetic groups: three in the ELN Favorable, one in the ELN Intermediate-I, one in the ELN Intermediate-II, and three in the ELN Adverse group. Among the other molecular markers, only NPM1 mutations were significantly associated with DNMT3A mutations (P=.004). The complete remission (CR) rate for the entire cohort was 41% (19/46, 95% CI: 27% to 57%). Six of eight DNMT3A-mutated pts (75%) reached CR, compared to 13 of 38 with wild-type DNMT3A (34%; P=.05). All five pts with mutated DNMT3A and mutated NPM1 achieved CR, a significantly better response rate than that observed among the remaining pts (P=.008). The median overall survival of DNMT3A-mutated pts was longer than that of DNMT3A-wild-type pts (16.8 vs 11.0 months), but the difference did not reach statistical significance, likely due to the relatively small number of pts. Our group has shown that high levels of microRNA miR-29b, which targets and downregulates DNMT3A, are significantly associated with response to decitabine. (Blum W et al, PNAS 107:7473) Among the pts achieving CR, DNMT3A mutations were present in the pts with the lowest miR-29b expression levels (Wilcoxon’s P=.02 for the comparison of miR-29b expression between CR pts with and without DNMT3A mutations). Indeed, the miR-29b expression levels in these three pts was similar to that of pts who did not achieve CR, thereby supporting an impact of the mutation independent from that of the miR. These initial findings, while based on a small sample size, suggest a potential link between DNMT3A mutations and response to decitabine. If confirmed in a larger cohort, screening for DNMT3A mutation status could be used for risk-adapted stratification of AML pts to decitabine-based regimens.Supported by NIH/NCI grants NO1 CM62207; UO1 CA76576; K23CA120708 (WB); R01 CA102031 (GM), K12CA133250 (JCB, AW); AW is an ASH-AMFDP Scholar.Table 1:Pt Characteristics and Response to Decitabine TreatmentCR n=19No CR n=27PAge (years) median (range)72 (62 – 85)76 (32 – 84).09WBC (×103/μl) median (range)2.7 (1.1 – 119.5)2.2 (0.6 – 150).74Bone marrow blast count median (range)35 (20 – 87)39 (18 – 92).23ELN genetic category no..77favorable55intermediate-I45intermediate-II38adverse79DNMT3A mutated no.62.05NPM1 mutated no.63.13DNMT3A mutated &NPM1mutated no.50.008IDH1 or IDH2 mutated no.341.0TET2 mutated no.351.0FLT3-ITD no.21.56FLT3-TKD no.011.0CEBPA mutated no.14.39P values are from the Wilcoxon test for continuous data or Fisher's exact test for count data and not adjusted for multiple comparisons Disclosures:Off Label Use: Decitabine alone or in combination with bortezomib is not approved for the treatment of AML.

Mapping of Genetic Deletions on Chromosome 3 in Colorectal Cancer: Loss of 3p25-pter is Associated with Distant Metastasis and Poor Survival

There is no detailed analysis of loss of heterozygosity (LOH) on chromosome 3 in colorectal cancer (CRC). Our aim was to define frequently deleted loci on chromosome 3 and to explore novel prognostic markers and the locations of candidate tumor suppressor genes associated with CRC. LOH at 23 microsatellite markers spanning on chromosome 3 was determined in 112 sporadic CRC by automated fluorescence-based polymerase chain reaction. Genetic loss was assessed for the clinicopathological significance by univariate and multivariate analyses. Fifty-eight (51.8%) of 112 carcinomas exhibited LOH at one or more loci tested. Among seven loci with high LOH rates, allelic losses at D3S1297 and D3S1266 occurred more frequently in younger patients. A marked gender distortion for genetic deletion was observed at six loci, where LOH was identified more frequently in male cases. For clinical outcome, LOH solely at D3S1297 (3p26.3) was significantly associated with distant metastasis (P=0.001) and was indicative of a shorter overall survival (P=0.014). In addition, loss of one common deletion region at 3p25-pter was significantly correlated to distant metastasis (P=0.009) and had an adverse effect on patients' overall survival in univariate and multivariate tests (P=0.009 and 0.001, respectively). Loss of chromosome 3p25-pter could act as an independent predicator of poor prognosis in CRC, suggesting that microsatellite analysis is a useful means to stratify patients into different risk groups. In addition, inactivation of candidate tumor suppressor genes in this region might involve in CRC progression.

A Leishmania minicircle DNA footprint assay for sensitive detection and rapid speciation of clinical isolates

Diversity in clinical outcome, due to different species of Leishmania, and its presence in asymptomatic blood donors in endemic areas warrant development of methods that are sensitive and can rapidly identify infecting species. The kinetoplast minicircle DNA is known to have heterogeneity in sequence and is present in many thousands of copies in Leishmania. Fluorescence-based polymerase chain reaction (PCR) was used to amplify minicircle DNA from six Leishmania species from different geographic locations. The sequences were then used to construct a phylogenetic tree. Speciation of 46 blinded parasite clinical isolates from various geographic regions was validated using the assay. Analysis displayed a distinct cluster for each species or strain. Forty-three of 46 isolates were correctly assigned to the same species identified by isoenzyme electrophoresis. The three untyped isolates were all either new species or samples from a unique geographic region. The minicircles of the three isolates formed new clusters in the tree analysis. Using minicircle DNA as PCR target, the sensitivity of the parasite detection in the spiked blood samples was five parasites per mL. Increased sensitivity and speciation without the need for parasite culture will be useful for diagnosis and treatment in clinical settings.

Interleukin-1β gene in esophageal, gastric and colorectal carcinomas

Interleukin (IL)-1 gene polymorphisms are associated with development of gastric atrophy and with increased risk of gastric carcinoma. A -31C to T base transition in the promoter region of this gene is involved in carcinogenic changes within the stomach, especially in Helicobacter pylori infected individuals. We examined association between IL-1 locus polymorphisms and risk of esophageal, gastric and colorectal carcinomas in Japanese patients with H. pylori infection. IL-1B and IL-1RN polymorphisms were analyzed in 136 controls, 75 patients with esophageal carcinoma, 186 patients with gastric carcinoma, 69 patients with colorectal carcinoma, and 18 patients with ulcerative colitis (UC). For IL-1B-511 and -31 polymorphisms were determined by fluorescence-based polymerase chain reaction single-strand conformation polymorphism analysis. For IL-1 receptor antagonist gene (IL-1RN), penta-allelic variable number of tandem repeats (VNTR) was determined by PCR-standard agarose gel electrophoresis. For gastric carcinoma, IL-1B-511 heterozygotes (OR, 0.48; 95% CI, 0.3-0.9; p=0.0115) and T carriers (OR, 0.52; 95% CI, 0.3-1.0; p=0.0185) had a significantly reduced risk of carcinoma. For colorectal carcinoma, IL-1B-511 heterozygotes (OR, 0.34; 95% CI, 0.2-0.7; p=0.0028) and T carriers (OR, 0.43; 95% CI, 0.2-0.9; p=0.0015) had a significantly low risk of carcinoma. No significant difference was observed in the frequencies of IL-1B-31C/T and IL-1RN genotypes between controls and the esophageal carcinoma patients. Our results shows that IL-1B-511C/T and T carrier state may indicate less risk for gastric and colorectal carcinoma in the Japanese population.

Detection of Mutant BRAF Alleles in the Plasma of Patients with Metastatic Melanoma

Mutations in the BRAF oncogene at amino acid 600 have been reported in 40 to 70% of human metastatic melanoma tissues, and the critical role of BRAF in the biology of melanoma has been established. Sampling the blood compartment to detect the mutational status of a solid tumor represents a highly innovative advance in cancer medicine, and such an approach could have advantages over tissue-based techniques. We report the development of a fluorescence-based polymerase chain reaction (PCR) assay to detect mutant BRAF alleles in plasma. A mutant-specific PCR assay was optimized to specifically amplify the mutant BRAF allele without amplifying the wild-type allele. Experiments mixing DNA from a BRAF mutant melanoma cell line with wild-type human placental DNA in varying proportions were performed to determine the threshold of this assay and to compare it with routine DNA sequencing. The assay was then applied to tissue and plasma specimens from patients with metastatic melanoma. The assay detected 0.1 ng of mutant DNA mixed in 100 ng of wild-type DNA and was 500-fold more sensitive than DNA sequencing. The assay detected mutant BRAF alleles in plasma samples from 14 of 26 (54%) metastatic melanoma patients. These data demonstrate the feasibility of blood-based testing for BRAF mutations in metastatic melanoma patients.

Factor V Leiden Does Not Affect Bleeding in Aprotinin Recipients after Cardiopulmonary Bypass

Carriers of the factor V Leiden mutation (FVL) are resistant to activated protein C proteolysis. Therefore, they are at increased risk of thromboembolic events. Aprotinin is an unspecific proteinase inhibitor frequently used during cardiac surgery procedures to reduce bleeding. However, aprotinin may cause thromboembolic complications after cardiopulmonary bypass (CPB). The primary endpoint of this study was the amount of blood loss after CPB in aprotinin recipients, and secondary endpoints were thromboembolic complications. A total of 1,447 consecutive patients who underwent cardiac surgery with CPB were prospectively enrolled. All patients were screened for FVL by a fluorescence-based polymerase chain reaction method. Linear and logistic regression analyses were performed to assess associations of FVL on bleeding and thromboembolic complications. One hundred seven individuals (7.4%) were heterozygous FVL carriers. No difference was found between FVL carriers and noncarriers regarding age, sex, CPB, type of operation, EuroSCORE, antiplatelet treatment, and reoperation. FVL was not significantly associated with postoperative blood loss, whereas a significant influence was found for female sex (P < 0.0001), duration of CPB (P < 0.0001), reoperation (P = 0.001), and preoperative antiplatelet treatment (P < 0.002). Multiple linear regression analysis for total blood loss had an observed power of at least 99%. FVL carriers faced the same risk for postoperative transfusion (P = 0.391), reoperation (P = 0.675), myocardial infarction (P = 0.44), stroke (P = 0.701), and 30-day mortality (P = 0.4) as did noncarriers. These data suggest that FVL carriers do not have reduced blood loss compared with noncarriers. Furthermore, the combination of aprotinin and FVL does not enhance the risk for thromboembolic complications.

Interleukin 10.G microsatellite in the promoter region of the interleukin-10 gene in severe sepsis

The highly polymorphic interleukin 10.G (IL10.G) microsatellite located in the promoter region of the interleukin-10 (IL-10) gene exerts a positive transcriptional regulatory effect on IL-10 gene expression and correlates with the in vitro IL-10 secretion. This study was conducted to investigate whether IL10.G microsatellite is associated with the incidence and/or the outcome of severe sepsis. One hundred and fifteen patients with severe sepsis who had been treated at the intensive care unit of the university hospital were studied. One hundred and forty-one healthy individuals served as controls. IL10.G microsatellite genotyping was performed with the following two methods: fluorescent based polymerase chain reaction (PCR) techniques and silver staining of the amplified DNA fragment in polyacrylamide gel. Alleles were defined according to the size of the amplified DNA product. Ten alleles and 36 genotypes were detected both in the patients with severe sepsis and in the healthy controls. Allele IL10.G9 and allele IL10.G13 were the commonest alleles with the frequencies of 32.6% and 21.3% respectively in the patients with severe sepsis, and 34% and 27% respectively in the healthy controls. The allele frequencies of IL10.G microsatellite were neither different between the patients with severe sepsis and the healthy controls (P > 0.05), nor between survivors and non-survivors (P > 0.05). However, the frequency of one common allele IL10.G13 was slightly lower in the patients with severe sepsis than in the healthy controls (21.3% vs 27%, P > 0.05), and the frequency of allele IL10.G9 was slightly higher in the non-survivors than in the survivors (37.1% vs 28.1%, P > 0.05). IL10.G microsatellite may neither contribute to the susceptibility to severe sepsis nor to the fatal outcome of severe sepsis.

Microsatellite analysis of the APC gene and immunoexpression of E-cadherin, catenin, and tubulin in esophageal squamous cell carcinoma

The histological features that accompany the development and progression of solid tumors are known to be controlled by a distinct cascade of molecular events. One such event is the inactivation of tumor suppressor genes, such as the adenomatous polyposis coli (APC) gene. Disruption of the cadherin-catenin cell adhesion complex also plays a role in the initial steps of cancer invasion and metastasis whereas alterations in cell structural molecules, such as tubulin, may contribute to the cancer phenotype. The understanding of the status of these molecules in ESSC may provide novel markers that could impact on management of the disease. The present study examined alterations in the microsatellite sequence of the APC gene via fluorescent-based polymerase chain reaction in 100 cases of primary esophageal squamous cell carcinoma. In addition, the expression of E-cadherin, alpha- and beta-catenin, and alpha- and beta-tubulin was analyzed using immunohistochemistry. These data were then statistically compared with each other as well as the relevant clinicopathologic data. Although the APC markers (D5S210, D5S346, D5S299, and D5S82) tested did show an overall high frequency of allelic imbalance/loss of heterozygosity (62.48%) and microsatellite instability (41.27%), they did not show prognostic significance in the study cohort and were not correlated with the immunohistochemical data. The tubulin proteins showed no significant change in expression in the tumor tissue The decreased immunoreactivity of E-cadherin was statistically correlated with the presence of lymph node metastases (P = .0180). Although alpha- and beta-catenin as well as E-cadherin showed no direct prognostic value, E-cadherin may warrant further investigation as an indirect prognostic indicator by allowing more accurate prediction of lymph node metastases.

PCR Microchip Array Based on Polymer Bonding Technique

A polymerase chain reaction (PCR) microchip array with a dimension of 21.3×17.5 mm2 has been fabricated by the silicon micromachining technique. The chip is composed of 192 rectangular reaction chambers with the volume of 50 nL. In order to package the silicon-based PCR chip, a low-temperature bonding technique using polymers as the intermediate layers has been developed. The tested polymers include positive photoresist, polyimide, and epoxy. Using stamping and sandwich techniques, void-free and liquid-proof bondings of silicon to a glass cover were achieved with the three polymers. The experimental results of fluorescence-based PCR demonstrate that the DNA amplification can be performed in the chip arrays with nanoliter volumes and the polymer bonding technique is biocompatible and suitable for the microchip packaging.