Detection Of MC Research Articles

A DNA Polymerase Variant Senses the Epigenetic Marker 5-Methylcytosine by Increased Misincorporation.

Dysregulation of DNA methylation is associated with human disease, particularly cancer, and the assessment of aberrant methylation patterns holds great promise for clinical diagnostics. However, DNA polymerases do not effectively discriminate between processing 5-methylcytosine (5 mC) and unmethylated cytosine, resulting in the silencing of methylation information during amplification or sequencing. As a result, current detection methods require multi-step DNA conversion treatments or careful analysis of sequencing data to decipher individual 5 mC bases. To overcome these challenges, we propose a novel DNA polymerase-mediated 5 mC detection approach. Here, we describe the engineering of a thermostable DNA polymerase variant derived from Thermus aquaticus with altered fidelity towards 5 mC. Using a screening-based evolutionary approach, we have identified a DNA polymerase that exhibits increased misincorporation towards 5 mC during DNA synthesis. This DNA polymerase generates mutation signatures at methylated CpG sites, allowing direct detection of 5 mC by reading an increased error rate after sequencing without prior treatment of the sample DNA.

High Frequency of Both, Short and Long-Term Post-Transfusion Microchimerism Among the Regularly-Transfused Thalassemic Patient Population

Background: Transfusion of blood products has been associated with the long term engraftment of donor cell-derived genetic material in the recipient (microchimerism: MC), whose clinical implications have not been sufficiently studied. The manifestation of post-transfusion MC has mainly been studied among severe trauma injured patients, heavily transfused at short term periods of time.Aim: We investigated the presence of post-transfusion MC among the regularly-transfused thalassemic patient population of Western Greece, and we analyzed factors, which might influence the frequency and severity of this phenomenon.Patients - Methods: Sixty-four transfusion-dependent homozygous β-thalassemic patients [female (N=32), male (N=32), with a median age of 42 years (range 22-60 years), completely transfusion-dependent (N=54) or partially-dependent (n=10) and 21 never-transfused male controls (median age 41 years, range 24-53 years) were studied. To reveal the presence of MC we have used a real-time PCR technique with SYBR Green, for the detection of 12 HLA-DR alleles. To enhance the ascertainment of MC we used primers corresponding to 12 loci among eight somatic chromosomes that have relatively common, phenotypically silent biallelic insertion or deletion polymorphisms (SO-1 through SO-11). All reactions were performed in double and positive samples were re-tested in double. Overall, 205 samples were tested, and samples from 57 patients were re-tested, after a median of 9.5 months. Moreover, a group of 21 patients, randomly selected, was tested for a third time, after a median of 17.6 months from the second test and 26.4 months from the baseline test.Results: Fifty-one patients (79.7%) and 8 controls (38%) exhibited a positive reaction to 1-7 chimeric alleles (patients) and 1 or 2 alleles (controls). One patient could not identified. Twenty patients (31.2%) and 6 controls (28.5%) have shown positivity for 1 chimeric alleles. Sixteen patients (25%) and 2 controls (9.5%) have shown positivity for 2 chimeric alleles. All the other positive patients but no control subject, had a positive signal for 3 or more alleles. Overall, 21 patients had evidence of long-term MC, and 8 patients only short-term MC, whereas 22 patients detected with both, short- and long-term MC. The detection of positivity in the control subjects could be interpreted as persistence of feto-maternal MC. As expected, there was a female predominance in MC detection, with higher frequency of positive samples (30/32 ~ 93.7% vs 22/32 ~ 68.7%, p<0.01) and higher mean number of positive alleles per subject, compared to males (x±SD: 2.46±1.69 vs 1.15±0.97, p=0.0004). Patients younger than 44 years had lower number of positive signals (1.06±1.5 vs 2.21±1.89, p=0.06), and the same was also true for patients with lower transfusion frequency (<6 units per year: 0.90±0.83), compared to moderately (6-20 units per year) or intensely transfused patients (>20 units per year: 2.0±1.63, p=0.045). Moreover, splenectomized patients had also higher mean number of chimeric alleles, compared to non-splenectomized (2.13±1.76 vs 1.39±1.01, p=0.054). Finally, in 7 of the 21 patients, which MC persisted at all 3 time points, we detected the gradual establishment of 6 new chimeric polymorphisms, but also the disappearance of 4 short-term chimeric polymorphisms, during a median period of 26.4 months.Conclusion: High frequency of regularly transfused thalassemic patients establish long-term microchimerism, which is an active and evolving phenomenon, related to transfusion burden and is influenced by patient's gender, age and splenectomy. The clinical impact of this phenomenon is as yet unknown and unpredicted. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Quenching photoluminescence of Carbon Quantum Dots for detecting and tracking the release of Minocycline

This work reports the preparation of Carbon Quantum Dots via one-step microwave-assisted synthesis, to be employed for the determination of Minocycline antibiotic. Linear relationship was observed between photoluminescence quenching of CQDs and Minocycline concentration between 0.28–30.39 μM. UV–vis and PL Spectroscopy revealed that PL decrease arise from Inner Filter Effect. Selectivity studies were conducted to evaluate interference of various compounds on MC detection. In addition, Minocycline was loaded on Chitosan-grafted-oligomeric Lactic Acid microspheres, to be used as a drug carrier of Minocycline. Thereafter, CQDs were used to determine loading capacity, encapsulation efficiency, and cumulative drug release of Minocycline-loaded microspheres. Optimum samples showed loading capacity between 14.1–26.344 %, and encapsulation efficiency between 34.48–64.37%. CHS-g-OLA2 sample showed diminished burst release, and 50 % cumulative drug release during 4 days. Mathematical modelling showed that CHS-g-OLA2 sample follows Higuchi kinetic model, which describes the release of drug from swellable matrix by diffusion.

Peripheral Blood Monoclonal B-Cell and/or Plasma Cell Detection By Flow Cytometry in Screening and Monitoring EBV-Positive Post-Transplant Lymphoproliferative Disorders

Background Post-transplant lymphoproliferative disorder (PTLD) is a serious complication that can occur following an allogenic hematopoietic stem cell transplant (allo-HSCT). PTLD occurs in approximately 0.8% to 20% of patients following an allo-HSCT and is associated with Epstein-Barr virus (EBV) infection in about 60% to 80% of patients. EBV positive (EBV+) PTLD generally arises early, several months following transplantation. The routine methods to diagnose EBV+ PTLD are clinical symptoms, EBV copy number, imageological examination, and pathological diagnosis, of which pathological diagnosis is the gold standard. Yet obtaining a biopsy is not possible with some patients and is not applicable for post-therapy monitoring. Therefore, there is an urgent need to find a simple, highly efficient, feasible, sensitive, and specific diagnostic and monitoring tool. Flow cytometry (FCM) has been established as a highly cost-effective method to diagnose lymphoma over the past several decades, and particularly for screening monoclonal B and/or plasma cells (MC B/P). Several recent publications as well as our own published and unpublished data have found that MC B/P are present in the peripheral blood of most PTLD patients. To this end, FCM detection of MC B/P in PB is a promising screening and monitoring method for EBV(+) PTLD. Objective To investigate the effectiveness of detecting MC B/P in PB by FCM for EBV+ PTLD screening and monitoring. Methods A total of 1470 patients received allo-HSCT at the Hebei Yanda Ludaopei Hospital, China from January 2018 through December 2019. We conducted a retrospective study of 481 patients with fever and lymphadenopathy following allo-HSCT. Patient PB was extracted for FCM MC B/P analysis. Plasma EBV viral loads were detected by PCR. The median fellow-up time was 6 months (range: 2 days to 21 months). The relationships of PB MC B/P, EBV load and clinical EBV-associated PTLD symptoms were investigated. Results: MC B/Ps were detected in the PB of 47 patients, of which 29 cases had monoclonal B cells, 14 cases had detectable co-existence of monoclonal B cells and monoclonal plasma cells, and 4 cases had detectable monoclonal plasma cells. The median time to PTLD onset following allo-HSCT was 70 days (range: 33 days to 491 days). The median percentage of monoclonal B cells was 0.43% (range: 0.1% to 23.41%. The median percentage of monoclonal plasma cells was 0.25%). Forty of 47 patients (85.1%) were finally diagnosed with PTLD using clinical comprehensive examination of symptoms. The incidence of clinical EBV+ PTLD was 2.9% (44 of 1470 cases). By FCM, MC B/Ps were observed in the PB of 91% of the patients (40 of 44 cases). No MC B/Ps were found in the PB of 4 patients. MC B/P detection in PB by FCM screening could effectively predict the incidence of EBV+ PTLD with a sensitivity of 90.91% and specificity of 98.40%. The positive predictive value was 85.17% and the negative predictive value was 99.08%. There was no significant correlation between EBV viral copy number and percentage of MC B/P. The median fellow-up time was 6 months (range: 2 days to 21 months) for all patients. The therapeutic response rate was 87.5% (35 of 40 patients) for MC B/P positive EBV+ PTLD patients. In those patients with a therapeutic response, clinical symptoms improved, lymph nodes significantly shrank, EBV viral loads decreased, MC B/Ps decreased or vanished, and CD20+ cell proportion decreased or vanished. Among the 7 patients who had MC B/Ps in PB but were not diagnosed with PTLD , their clinical symptoms recovered after treatment with immunosuppressive therapy. Conclusion By detecting PB MC B/Ps using FCM, we can successfully screen for EBV+ PTLD and monitor patient therapeutic responses in most cases. FCM to detect MC B/Ps in PB is a new, promising method for the diagnosis and monitoring of EBV+ PTLD. Compared to a biopsy, this appears to be a more simple, easier and more applicable tool to diagnose and monitor EBV+ PTLD. Disclosures No relevant conflicts of interest to declare.

Comparison of confocal laser scanning colorful fundus imaging and color fundus photography for detection of diabetic retinopathy

Objective To compare the imaging characteristics and detection of various types of lesions in diabetic retinopathy (DR) with colorful laser scanning fundus imaging (MSLI) and traditional color fundus photography (CFP). Methods Prospective case series observational study. A total of 38 eyes of 38 patients with DR diagnosed by clinical examination were included in the study. Among them, 21 were male and 17 were female; the mean age was 62.6±11.2 years; the average duration of diabetes was 14.3±7.5 years. All the patients were performed CFP, MSLI, frequency domain optical coherence tomography (SD-OCT), fluorescein angiography (FFA) examination. Using the Helielberg Spectralis HRA+OCT MSLI inspection, one scan simultaneously obtained 488 nm blue reflection (BR), 515 nm green light reflection (GR), 820 nm infrared light reflection (IR), and multicolor image (MC). The detection of traditional CFP and MC on microaneurysm (MA), hard exudation (HEX), cotton plaque (CWS), intraretinal hemorrhage (IRH), intraretinal microvascular abnormality (IRMA), venous bead (VB), venous ring (VL), macular edema (DME), macular anterior membrane (MEM) and laser photocoagulation (LB) were comparatively observed. The results of FFA examination were used as the diagnostic criteria for lesions. SD-OCT was used to determine the location and depth of lesions and the diagnostic reference for DME and MEM. Results The numbers of eyes with MA (χ2=10.460), DME (χ2=4.006), MEM (χ2=4.444) was significantly higher in MC than that of traditional CFP. But the number of eyes with IRH (χ2=0.103), CWS (χ2=1.515), HEX (χ2=0.227), IRMA (χ2=0.051), VB (χ2=0.001), VL (χ2=0.149), VH (χ2=0.693) and LB (χ2=0.720) were not statistically significant between two methods (P>0.05). The imaging quality of MSLI mode is obviously better than that of traditional CFP. Among them, GR imaging shows the best structural changes of superficial retina in MA, CWS, HEX, MEM, etc. IR imaging shows clear depth in deep retina such as LB. DME was green on MC and the weak low-reflection dark area was visible on the IR image, which were consistent with the DME range indicated by the SD-OCT examination. Conclusions Compared with the traditional CFP, the MSLI can clearly show the DR lesion. The number of checkouts is high on MA, DME and MEM by MC image. Key words: Diabetic retinopathy/diagnosis; Ophthalmoscopy; Microscopy, confocal; Tomography, optical coherence; Color fundus photography

DNA Photocrosslinking Using 3-Vinylcarbazole Derivatives in Two-color Detection of Methylcytosine

The methylated DNA base 5-methylcytosine (mC) is the most important factor in the epigenetic regulation of gene expression. 3-cyanovinylcarbazole nucleoside (CNVK) photocrosslinks with mC more rapidly than with cytosine (C). In the present study, we evaluated the effect of the substituents of various 3-vinylcarbazole derivatives on mC- and C-mediated DNA photocrosslinking for the quantitative detection of mC. We also demonstrated the quantitative detection of mC via photocrosslinking using 3-vinylcarbazole derivatives on a DNA chip. The results suggest that this photocrosslinking reaction has potential for the selective detection of mC in DNA strands via photocrosslinking using 3-cyanovinylcarbazole derivatives.

Near-set Based Mucin Segmentation in Histopathology Images for Detecting Mucinous Carcinoma.

This paper introducesnear-set based segmentation method for extraction and quantification of mucin regions for detecting mucinouscarcinoma (MC which is a sub type of Invasive ductal carcinoma (IDC)). From histology point of view, the presence of mucin is one of the indicators for detection of this carcinoma. In order to detect MC, the proposed method majorly includes pre-processing by colour correction, colour transformation followed by near-set based segmentation and post-processing for delineating only mucin regions from the histological images at 40×. The segmentation step works in two phases such as Learn and Run.In pre-processing step, white balance method is used for colour correction of microscopic images (RGB format). These images are transformed into HSI (Hue, Saturation, and Intensity) colour space and H-plane is extracted in order to get better visual separation of the different histological regions (background, mucin and tissue regions). Thereafter, histogram in H-plane is optimally partitioned to find set representation for each of the regions. In Learn phase, features of typical mucin pixel and unlabeled pixels are learnt in terms of coverage of observed sets in the sample space surrounding the pixel under consideration. On the other hand, in Run phase the unlabeled pixels are clustered as mucin and non-mucin based on its indiscernibilty with ideal mucin, i.e. their feature values differ within a tolerance limit. This experiment is performed for grade-I and grade-II of MC and hence percentage of average segmentation accuracy is achieved within confidence interval of [97.36 97.70] for extracting mucin areas. In addition, computation of percentage of mucin present in a histological image is provided for understanding the alteration of such diagnostic indicator in MC detection.

Pitfalls in microcystin extraction and recovery from human blood serum

BackgroundMicrocystins (MCs) contaminate water bodies due to cyanobacterial blooms all over the world, leading to frequent exposure of humans to MCs through consumption of meat, fish, seafood, blue-green algal products and water, accidental ingestion of contaminated water and scum during recreational activities and inhalation of cyanobacterial aerosols. For monitoring of human exposure, sensitive screening methods are needed. However, during the analytical process of various matrices, such as human serum, some problems appear to regularly occur during sample preparation and storage, leading to MC loss and thus to underestimation of the true MC concentration. The aim of the current study was therefore to assess the pitfalls of the MC-extraction method from human serum with more detail. MethodsSix MC congeners (MC-LR, -YR, -RR, -LA, -LW, -LF) and defined equimolar MC mixtures thereof were spiked into human serum, and quantified using the commercially available Adda-ELISA subsequent to standard extraction (methanol extraction with subsequent SPE). To detect the potential influence of sample storage and preparation/storage materials, different types of material such as glass, standard polypropylene and surface-treated polypropylene were compared. ResultsLoss of MC during preparation and storage is largely dependent on (1) the handling of the stored material, (2) the ‘surface’ of the storage material and (3) the hydrophobicity of the MCs. ConclusionsThe pitfalls described for MC analysis with the ELISA are primarily associated with sample preparation and clean-up and thus also apply to other analytical techniques for MC detection beyond the ELISA used. It can be concluded that ELISA-based methods are suitable tools for the detection of MCs in human sera and other samples.

Improving the carbon balance of fermentations by total carbon analyses

Carbon balancing of microbial fermentations is a valuable tool for the evaluation of the process performance and to identify the presence of undesired by-products. In this study, we demonstrate the relevance of total carbon (TC) analysis for carbon balancing in fermentations with the wild-type of Corynebacterium glutamicum by (i) quantifying significant amounts of dissolved inorganic carbonic species (TIC) in the culture medium and (ii) determining the effective (mass) carbon content of the biomass fraction (MC,X). In principle, TC based carbon balancing yielded at fully matching carbon balances. Thus, the application of our TC approach for the accurate detection of TIC and MC,X increased the total carbon recovery in standard batch fermentations with C. glutamicum on glucose from about 76% to carbon closures of 94–100% in contrast to conventional approaches. Besides, the origin of the missing 6%-gap could be attributed to incomplete quantification of all carbon sources in the liquid phase. To conclude this study, the concept of TC-based balancing was transferred to an l-lysine production process, successfully quantifying relevant system carbon fractions, which resulted in matched carbon recoveries.

Cardiac valve calcifications on low-dose unenhanced ungated chest computed tomography: inter-observer and inter-examination reliability, agreement and variability.

To determine inter-observer and inter-examination variability for aortic valve calcification (AVC) and mitral valve and annulus calcification (MC) in low-dose unenhanced ungated lung cancer screening chest computed tomography (CT). We included 578 lung cancer screening trial participants who were examined by CT twice within 3 months to follow indeterminate pulmonary nodules. On these CTs, AVC and MC were measured in cubic millimetres. One hundred CTs were examined by five observers to determine the inter-observer variability. Reliability was assessed by kappa statistics (κ) and intra-class correlation coefficients (ICCs). Variability was expressed as the mean difference ± standard deviation (SD). Inter-examination reliability was excellent for AVC (κ = 0.94, ICC = 0.96) and MC (κ = 0.95, ICC = 0.90). Inter-examination variability was 12.7 ± 118.2 mm(3) for AVC and 31.5 ± 219.2 mm(3) for MC. Inter-observer reliability ranged from κ = 0.68 to κ = 0.92 for AVC and from κ = 0.20 to κ = 0.66 for MC. Inter-observer ICC was 0.94 for AVC and ranged from 0.56 to 0.97 for MC. Inter-observer variability ranged from -30.5 ± 252.0 mm(3) to 84.0 ± 240.5 mm(3) for AVC and from -95.2 ± 210.0 mm(3) to 303.7 ± 501.6 mm(3) for MC. AVC can be quantified with excellent reliability on ungated unenhanced low-dose chest CT, but manual detection of MC can be subject to substantial inter-observer variability. Lung cancer screening CT may be used for detection and quantification of cardiac valve calcifications. • Low-dose unenhanced ungated chest computed tomography can detect cardiac valve calcifications. • However, calcified cardiac valves are not reported by most radiologists. • Inter-observer and inter-examination variability of aortic valve calcifications is sufficient for longitudinal studies. • Volumetric measurement variability of mitral valve and annulus calcifications is substantial.

Programmable and Highly Resolved In Vitro Detection of 5‐Methylcytosine by TALEs

Gene expression is extensively regulated by specific patterns of genomic 5-methylcytosine (mC), but the ability to directly detect this modification at user-defined genomic loci is limited. One reason is the lack of molecules that discriminate between mC and cytosine (C) and at the same time provide inherent, programmable sequence-selectivity. Programmable transcription-activator-like effectors (TALEs) have been observed to exhibit mC-sensitivity in vivo, but to only a limited extent in vitro. We report an mC-detection assay based on TALE control of DNA replication that displays unexpectedly strong mC-discrimination ability in vitro. The status and level of mC modification at single positions in oligonucleotides can be determined unambiguously by this assay, independently of the overall target sequence. Moreover, discrimination is reliably observed for positions bound by N-terminal and central regions of TALEs. This indicates the wide scope and robustness of the approach for highly resolved mC detection and enabled the detection of a single mC in a large, eukaryotic genome.

Meeting report of the First Symposium on Chimerism

The incidence and potentially different biological impact of naturally acquired maternal and fetal cells were enthusiastically and extensively discussed when basic scientists and clinicians met at the Medical University of Graz in Austria for an International Symposium on Chimerism (Fig. 1). The two day program featured presentations by speakers from 4 different continents, intermingled with short presentations focusing on technical aspects of chimerism analysis by representatives from the companies who sponsored the meeting. Details on the program and speakers list are available at the meeting’s website (http://chimerism.medunigraz.at/cms/website.php). As also exemplified by the various different types of studies published in the recently released journal Chimerism, this field is slowly but steadily expanding as new research teams working outside and within the transplantation field have become interested in the biologically intriguing phenomena of chimerism and microchimerism (Mc). The highlights of the meeting are summarized in this meeting report. Figure 1. The Symposium on Chimerism. Photo courtesy of Harry Schiffer, Copyright Graz Tourism. Technical Aspects of Chimerism Analysis Since the 1979 paper by Herzenberg and colleagues reporting enrichment of cells from pregnant women presumed to be fetal using flow-sorting techniques,1 many different studies have been published describing fetal or maternal cells in diverse hematopoietic cell types or tissues and organs. During the past decade, major technical improvements regarding the detection of these generally rare cells have been made. This has resulted in a shift from the more traditional XY fluorescent in situ hybridization (FISH) staining technique applied to tissue sections to more refined technologies such as real-time PCR for the detection of male-specific DNA sequences in genomic DNA extracted from peripheral blood cells or processed tissues. Real-time PCR reactions for mismatched HLA alleles as well as for other genetic markers (insertion/deletion) have been developed in the laboratories of meeting participants Dr Nelson (Fred Hutchinson Cancer Research Center, Seattle, USA), Dr Lambert (INSERM UMRs 1097, Marseille, France) and Dr van Halteren (Leiden University Medical Center, Leiden, the Netherlands). Unfortunately, many researchers are faced by the problem that this approach requires the availability of DNA from mother or offspring in order to find informative genetic markers. Of note, the newly developed QuantiChimera platform established at the laboratory of Dr Lambert can be contacted for technical support regarding state-of-the art PCR technology for Mc detection. Particularly relevant for investigators who are restricted to using FISH techniques for the identification of rare maternal or fetal chimeric cells is the collective observation that this technique does not always give consistent staining results. This may be explained by the fact that archived tissue samples are often subjected to different fixation protocols. As discussed by Dr Sedlmayr and Dr Kroneis (Medical University, Graz, Austria), FISH by itself is a less than optimal technique for the analysis of rare microchimeric cells given the frequency of false positive cells. This issue can in part be addressed by using two reverse sets of differently labeled X and Y probes. Also biochemical markers such as unique cell surface molecules or markers that can be used to distinguish fetal cells from adult cells are not perfectly reliable in the setting of analysis of extremely rare cells. Unambiguous identification of individual microchimeric cells can be done by combining either FISH analysis or analysis of biochemical markers with DNA genotyping of candidate cells isolated by laser microdissection.2 The introduction of a whole genome amplification step subsequently even allows combining DNA genotyping of single microchimeric cells with sequencing or comparative genome hybridization.3This exciting new approach is expected to further boost the chimerism field.

A nanosensor based on quantum-dot haptens for rapid, on-site immunoassay of cyanotoxin in environmental water

A nanoprobe based on quantum-dot (QD) haptens was synthesized by conjugating carboxyl quantum dots with aminoethyl-microcystin (MC)–leucine–arginine (LR). A two-alkyl group was introduced to supply a spacer between the QD nanoprobe and anti-MC–LR antibody to reduce the steric hindrances of immunoreaction. The sensor system based on a portable optofluidic platform exhibited a liner range of 0.10–4.0µg/L for MC–LR with a detection limit of 0.03µg/L. The proposed sensor has potential application in the rapid, on-site detection of MC–LR in real water samples.

Quick, Selective and Reversible Photocrosslinking Reaction between 5-Methylcytosine and 3-Cyanovinylcarbazole in DNA Double Strand

Selective photocrosslinking reaction between 3-cyanovinylcarbazole nucleoside (CNVK) and 5-methylcytosine (mC), which is known as epigenetic modification in genomic DNA, was developed. The reaction was completely finished within 5 s of 366 nm irradiation, and the rate of this photocrosslinking reaction was ca. 30-fold higher than that in the case of unmodified normal cytosine. There were no significant differences in the thermodynamic parameters and the kinetics of hybrid formation of oligonucleotide (ODN) containing CNVK and its complementary ODN containing C or mC at the photocrosslinking site, and suggesting that the quick and selective photoreaction has potential for the selective detection of mC in the DNA strand via the photocrosslinking reaction.

Electrochemical DNA Methylation Detection for Enzymatically Digested CpG Oligonucleotides

We describe the electrochemical detection of DNA methylation through the direct oxidation of both 5-methylcytosine (mC) and cytosine (C) in 5'-CG-3' sequence (CpG) oligonucleotides using a sputtered nanocarbon film electrode after digesting a longer CpG oligonucleotide with endonuclease P1. Direct electrochemistry of the longer CpG oligonucleotides was insufficient for obtaining the oxidation currents of these bases because the CG rich sequence inhibited the direct oxidation of each base in the longer CpG oligonucleotides, owing to the conformational structure and its very low diffusion coefficient. To detect C methylation with better quantitativity and sensitivity in the relatively long CpG oligonucleotides, we successfully used an endonuclease P1 to digest the target CpG oligonucleotide and yield an identical mononucleotide 2'-deoxyribonucleoside 5'-monophosphate (5'-dNMP). Compared with results obtained without P1 treatment, we achieved 4.4 times higher sensitivity and a wider concentration range for mC detection with a resolution capable of detecting a subtle methylated cytosine difference in the CpG oligonucleotides (60mer).

An 18-year Follow-up of Allergy Development Related to Nasal Metachromatic Cell Findings During Infancy

The ability to predict the development of allergic diseases in infants is important. Predictive biomarkers are wanted to improve the risk evaluation in addition to known heredity of allergy. Biomarkers taken during infancy need to be evaluated through longitudinal studies into adulthood. The objective of this study was to analyse the occurrence of metachromatic cells in the nasal mucosa during infancy (MC(infancy)) and evaluate the cells as predictive biomarkers of allergy development. Previously, MC(infancy) occurrences were analysed in 64 infants with and without allergy heredity, and related to allergy development at 18 months and 6 years of age. In this third follow-up at 18 years of age, current allergy symptoms were analysed. MC(infancy) findings were related to the cumulative number of allergic subjects. The predictive values of MC(infancy) and known heredity were compared. The cumulative number of subjects with allergy was 46, probable allergy 5, and no allergy 13. Detected MC(infancy) predicted allergy with high accuracy (31/33), but negative MC(infancy) findings did not exclude the risk (15/31). In the group of allergic subjects positive MC(infancy) were found in 31/46 (67%), positive heredity in 37/46 (80%) and one/both factors positive in 43/46 (93%). Detection of MC(infancy) could precede the debut of allergy symptoms by many years. Detected MC(infancy) predicted allergy development, but absence of MC(infancy) did not exclude the risk, and therefore this biomarker was not found to be adequate. There is a further need to find biomarkers with high ability to both predict and exclude the risk.

Fluorometric Identification of 5-Methylcytosine Modification in DNA: Combination of Photosensitized Oxidation and Invasive Cleavage

An efficient fluorometric detection system of DNA methylation has been developed by a combination of a photooxidative DNA cleavage reaction with 2-methyl-1,4-naphthoquinone (NQ) chromophore and an invasive cleavage reaction with human Flap endonuclease-1. Enzymatic treatment of a mixture of photochemically fragmented target oligodeoxynucleotides (ODNs) at 5-methylcytosine mC) and hairpin-like probe oligomer possessing a fluorophore (F) and a quencher (D) resulted in a dramatic enhancement of fluorescence. In contrast, fluorescence emission for the ODN containing cytosine but not mC at the target sequence was extremely weak. In addition, by monitoring the fluorescence change, this system allows for the detection of mC in DNA at subfemtomole amounts. This system would provide a highly sensitive protocol for determining the methylation status in DNA by fluorescence emission.

Site-specific discrimination of Cytosine and 5-methylcytosine in duplex DNA by Peptide nucleic acids.

For site-specific discrimination of cytosine (C) and 5-methylcytosine (mC) in duplex DNA, we developed a new method using peptide nucleic acids (PNAs). The combination of a PNA-assisted DNA displacement complex and a fluorescein-labeled probe oligomer allowed the detection of mC at the defined sites in target DNA using a restriction enzyme. After treatment of the complex with a restriction enzyme, strong fluorescence emission was observed for the complex containing C at the target site, whereas the fluorescence intensity for the complex containing mC was extremely weak.

Persistent donor chimaerism is consistent with disease-free survival following BMT for chronic myeloid leukaemia.

Chronic myeloid leukaemia (CML) can be treated successfully with allogeneic bone marrow transplantation (BMT) leading to long-term disease-free survival. Leukemia relapse, however, remains a significant clinical problem. Relapse following BMT presumably results from the expansion of small numbers of recipient leukaemic cells which have survived the conditioning therapy. In order to define patients who are at a high risk of leukaemia relapse, a variety of techniques have been employed to detect persistence of host haemopoiesis (mixed chimaerism, MC) or residual leukaemia (minimal residual disease, MRD). However, the precise relationship between the detection of MC and MRD post-BMT is unknown. We have investigated chimaerism and MRD status in 22 patients who were in clinical and haematological remission post-allogeneic BMT for chronic phase CML. Chimaerism was assessed using short tandem repeat PCR (STR-PCR) while BCR-ABL mRNA detection using reverse transcriptase polymerase chain reaction (RT-PCR) was performed to detect the presence of MRD. Seventeen patients received unmanipulated marrow (non-TCD) while in five patients a T cell-depleted transplant (TCD) was performed as additional GVHD prophylaxis. Chimaerism was evaluated in 18 patients (14 non-TCD, four TCD). Mixed chimaerism was an uncommon finding in recipients of unmanipulated BMT (21%) when compared to TCD BMT (100%). No evidence of MRD, as identified using the BCR-ABL mRNA RT-PCR assay, was detected in those patients who were donor chimaeras. Early and transient MC and MRD was detected in four patients (two non-TCD, two TCD) who have subsequently converted to a donor profile. One patient has stable low-level MC but remains MRD negative 4 years post-BMT. Late MC and MRD was observed in two patients who relapsed >6 years after TCD BMT for CML. We conclude that mixed chimaerism is a rare event in recipients of unmanipulated BMT and that donor chimaerism as detected by STR-PCR assay is consistent with disease-free survival and identifies patients with a low risk of leukaemic relapse post-BMT for CML.

Use of immunoglobulin heavy-chain and light-chain measurements in a multicenter trial to investigate monoclonal components: I. Detection

We assessed the combined use of serum protein electrophoresis (SPE) and nephelometric measurement of immunoglobulin heavy- and light-chain components for detecting serum monoclonal immunoglobulins (monoclonal components, MC) in 4788 unselected samples from 4173 patients. MC were detected in 514 samples from 390 patients. In 356 these were detected by SPE; the other 34 had a normal SPE pattern but an abnormal kappa:lambda light-chain ratio (KLR). Only 208 of the 356 (58%) samples with bands by SPE had abnormal KLRs. Samples with MC concentrations greater than 5 g/L had a higher proportion of abnormal KLRs (75%) than those with concentrations less than 5 g/L (42%). The KLR was abnormal in 13% of samples in which no MC were visible by SPE or immunofixation electrophoresis (IFE). Compared with quantitative measurements of immunoglobulin heavy and light chains, high-quality SPE remains the method of choice for the detection of MC. Quantitative methods, however, are able to detect additional MC, especially those containing free light chains, and in the absence of SPE and IFE will detect about 75% of MC present at greater than 5 g/L.