Real-time CHF detection from ECG signals using a novel discretization method

This study proposes a new method, equal frequency in amplitude and equal width in time (EFiA-EWiT) discretization, to discriminate between congestive heart failure (CHF) and normal sinus rhythm (NSR) patterns in ECG signals. The ECG unit pattern concept was introduced to represent the standard RR interval, and our method extracted certain features from the unit patterns to classify by a primitive classifier. The proposed method was tested on two classification experiments by using ECG records in Physiobank databases and the results were compared to those from several previous studies. In the first experiment, an off-line classification was performed with unit patterns selected from long ECG segments. The method was also used to detect CHF by real-time ECG waveform analysis. In addition to demonstrating the success of the proposed method, the results showed that some unit patterns in a long ECG segment from a heart patient were more suggestive of disease than the others. These results indicate that the proposed approach merits additional research.     

A new detection method for ATRX gene mutations using a mismatch-specific endonuclease

X-linked alpha-thalassemia/mental retardation syndrome (ATR-X, OMIM 301040) is a syndromic form of X-linked mental retardation (XLMR). It is caused by a mutation in the ATRX gene, which is also involved in other syndromic forms of XLMR as well as in non-syndromic XLMR, both in males and in females. To analyze the full range of disease-causing mutations for genetic counseling and to establish phenotype-genotype correlations, we have established a new screening method for mutations in the ATRX gene, which uses mismatch-specific endonuclease. We applied this method to confirm 13 known mutations in our patients, some of which have been difficult to be demonstrated by conventional denaturing high-performance liquid chromatography. Furthermore, we found four additional mutations in four ATR-X patients whose clinical diagnosis had not been confirmed at the molecular level. In this method, experimental conditions do not need to be altered depending on mutation sites, and it should be the alternative method for mutation screening.     

A novel method for retinal optic disc detection using bat meta-heuristic algorithm

Normally, the optic disc detection of retinal images is useful during the treatment of glaucoma and diabetic retinopathy. In this paper, the novel preprocessing of a retinal image with a bat algorithm (BA) optimization is proposed to detect the optic disc of the retinal image. As the optic disk is a bright area and the vessels that emerge from it are dark, these facts lead to the selected segments being regions with a great diversity of intensity, which does not usually happen in pathological regions. First, in the preprocessing stage, the image is fully converted into a gray image using a gray scale conversion, and then morphological operations are implemented in order to remove dark elements such as blood vessels, from the images. In the next stage, a bat algorithm (BA) is used to find the optimum threshold value for the optic disc location. In order to improve the accuracy and to obtain the best result for the segmented optic disc, the ellipse fitting approach was used in the last stage to enhance and smooth the segmented optic disc boundary region. The ellipse fitting is carried out using the least square distance approach. The efficiency of the proposed method was tested on six publicly available datasets, MESSIDOR, DRIVE, DIARETDB1, DIARETDB0, STARE, and DRIONS-DB. The optic disc segmentation average overlaps and accuracy was in the range of 78.5–88.2% and 96.6–99.91% in these six databases. The optic disk of the retinal images was segmented in less than 2.1 s per image. The use of the proposed method improved the optic disc segmentation results for healthy and pathological retinal images in a low computation time.

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Graphical abstract ?

     

A new method for the detection of Pneumocystis jirovecii using flow cytometry

Pneumocystis jirovecii is an opportunistic pathogen responsible for severe pneumonia in immunocompromised patients. Its diagnosis has been based upon direct microscopy either by classic staining (Gomori Grocott) or by epifluorescence microscopy (immunofluorescence staining, IFS), both of which are time-consuming and low on sensitivity. Our aim was to develop a flow cytometric (FC) protocol for the detection of P. jirovecii on respiratory samples. In our study, 420 respiratory samples were analysed in parallel by IFS and FC, and compared from clinical diagnosis to its resolution upon specific anti-Pneumocystis therapy. The optimum specific antibody concentration for FC analysis was determined to be 10 μg/ml, without any cross-reactions to bacteria or fungi. All positive cases detected by IFS were positive by FC; however, FC classified eight samples to be positive which were classified as negative by routine technique. These samples were obtained from patients with respiratory symptoms who responded favourably to Pneumocystis-specific therapy and were subsequently considered to be true-positives. Using clinical diagnosis as a reference method, FC showed 100% sensitivity and specificity, whereas IFS showed 90.9% sensitivity and 100% specificity. According to our results, a new diagnostic approach is now available to detect P. jirovecii in respiratory samples.     

A novel mitochondria-targeting method using special staining for the detection of apoptotic hepatocytes

ABSTRACT

Early detection of apoptotic cells on histological slides is of major importance for both diagnostic and research areas. In the current study, the aim was to propose a convenient method to stain the mitochondria and establish whether hepatocytes undergoing apoptosis can be identified in tissue sections using the proposed method. Liver tissue from five adult chinchillas was fixed with 10% neutral buffered formalin for Goldner’s trichrome (GT) and Groat’s iron hematoxylin and eosin (HE) stains and with Kolster’s fixative for the Heidenhain’s iron hematoxylin procedure. The HE and GT-stained sections showed the morphological features consistent with apoptosis i.e., homogenous intensely acidophilic cytoplasm, cell shrinkage with an irregular outline, nuclear shrinkage with cloudy karyoplasm, and karyopyknosis in the late stage. Sections stained with Heidenhain’s iron hematoxylin method was used to pinpoint mitochondria and revealed cells which were undergoing the first stages of the apoptosis process i.e., disappearance of mitochondria from the cell, chromatin condensation and margination, paracentral localization of nucleoli, and vacuolated nuclei. In more advanced stages of apoptosis, cells presented significant nuclear and cytoplasmic changes. It was concluded that this is the first report targeting the mitochondria, by performing inexpensive histological staining techniques, in order to assess dead cells in situ.     

UPD detection using homozygosity profiling with a SNP genotyping microarray

Single nucleotide polymorphism (SNP) based chromosome microarrays provide both a high‐density whole genome analysis of copy number and genotype. In the past 21 months we have analyzed over 13,000 samples primarily referred for developmental delay using the Affymetrix SNP/CN 6.0 version array platform. In addition to copy number, we have focused on the relative distribution of allele homozygosity (HZ) throughout the genome to confirm a strong association of uniparental disomy (UPD) with regions of isoallelism found in most confirmed cases of UPD. We sought to determine whether a long contiguous stretch of HZ (LCSH) greater than a threshold value found only in a single chromosome would correlate with UPD of that chromosome. Nine confirmed UPD cases were retrospectively analyzed with the array in the study, each showing the anticipated LCSH with the smallest 13.5 Mb in length. This length is well above the average longest run of HZ in a set of control patients and was then set as the prospective threshold for reporting possible UPD correlation. Ninety‐two cases qualified at that threshold, 46 of those had molecular UPD testing and 29 were positive. Including retrospective cases, 16 showed complete HZ across the chromosome, consistent with total isoUPD. The average size LCSH in the 19 cases that were not completely HZ was 46.3 Mb with a range of 13.5–127.8 Mb. Three patients showed only segmental UPD. Both the size and location of the LCSH are relevant to correlation with UPD. Further studies will continue to delineate an optimal threshold for LCSH/UPD correlation. © 2011 Wiley‐Liss, Inc.     

Development of an RT-PCR detection method for mud crab reovirus

Mud crab reovirus (MCRV) causes high mortality in the cultivated mud crab. To control better an outbreak of this virus, a rapid, specific and sensitive detection method based on RT-PCR was developed. The MCRV detection method was designed based on the one-step and two-step RT-PCR which resulted in the amplification of predicted products of 433 and 304 bp. The method is specific as no cross-reaction was observed between grass carp hemorrhage virus (GCHV), white spot syndrome virus (WSSV), tiger frog virus (TFV), infectious spleen and kidney necrosis virus (ISKNV), fish nervous necrosis virus (NNV) and the muscovy duck virus (MDRV). One-step PCR amplification could detect 10(-8) microg of purified MCRV dsRNA, while two-step PCR amplification could detect 10(-9) microg MCRV dsRNA. At an early stage of infection, MCRV could be detected in the heart, thoracic ganglion, muscle, intestine, gut, hemolymph, gonad and gill, but not in the stomach or hepatopancreas. However, in the moribund mud crabs, MCRV could be detected in all tissues examined.     

A novel detection method for Alaria alata mesocercariae in meat

Distomum musculorum suis (DMS), the mesocercarial stage of the trematode Alaria alata, can cause severe damages within their hosts, and since several reports about cases of human larval alariosis have been published, it became apparent that infected game animals and in particular wild boars are a potential source of infection for both humans and animals. A final statement concerning the health risks for consumers could not be given due to the lack of information about both the prevalence of DMS and the suitability of Trichinella inspection methods to detect this parasite in wild boar meat. Our studies concentrate on (1) the verification of suitability of the official digestion methods for Trichinella spp. for DMS detection in wild boars, (2) development, optimization, and validation of methods, and (3) the distribution of the parasites within their paratenic hosts. A total of 868 individual samples/digests from 48 wild boars were analyzed by the reference method for Trichinella detection in meat samples according to regulation (EC) No. 2075/2005. In addition to the official protocol, a method modification with Pankreatin© and bile acid was applied for analysis of adipose tissue samples (n?=?89). On the basis of our results, a new detection method based on a larvae migration technique was developed and used for detection of DMS in 574 single samples. Furthermore, the distribution patterns of DMS in wild boars in a total of 1377 single sample migrations/digestions from 35 positive animals were analyzed by application of all three methods. The official digestion method for Trichinella spp. in wild boars meat is inapplicable for the detection of A. alata mesocercariae as it shows shortcomings in both digestion and sampling. A direct comparison between the newly developed A. alata mesocercariae migration technique and the official digestion method for Trichinella spp. based on 574 single samples from 18 animals clearly shows that the sensitivity to detect A. alata developmental stages in tissues of wild boars of the new method is nearly 60% higher compared with the magnetic stirrer method for pooled sample digestion as laid down in regulation (EC) No. 2075/2005. Among other advantages, this method offers a simple, highly applicable, fast, and cost effective way to detect DMS in wild boars which is already applicable in routine veterinary inspection.     

AdnAB: a new DSB-resecting motor–nuclease from mycobacteria

The resection of DNA double-strand breaks (DSBs) in bacteria is a motor-driven process performed by a multisubunit helicase–nuclease complex: either an Escherichia coli-type RecBCD enzyme or a Bacillus-type AddAB enzyme. Here we identify mycobacterial AdnAB as the founder of a new family of heterodimeric helicase–nucleases with distinctive properties. The AdnA and AdnB subunits are each composed of an N-terminal UvrD-like motor domain and a C-terminal nuclease module. The AdnAB ATPase is triggered by dsDNA with free ends and the energy of ATP hydrolysis is coupled to DSB end resection by the AdnAB nuclease. The mycobacterial nonhomologous end-joining (NHEJ) protein Ku protects DSBs from resection by AdnAB. We find that AdnAB incises ssDNA by measuring the distance from the free 5′ end to dictate the sites of cleavage, which are predominantly 5 or 6 nucleotides (nt) from the 5′ end. The “molecular ruler” of AdnAB is regulated by ATP, which elicits an increase in ssDNA cleavage rate and a distal displacement of the cleavage sites 16–17 nt from the 5′ terminus. AdnAB is a dual nuclease with a clear division of labor between the subunits. Mutations in the nuclease active site of the AdnB subunit ablate the ATP-inducible cleavages; the corresponding changes in AdnA abolish ATP-independent cleavage. Complete suppression of DSB end resection requires simultaneous mutation of both subunit nucleases. The nuclease-null AdnAB is a helicase that unwinds linear plasmid DNA without degrading the displaced single strands. Mutations of the phosphohydrolase active site of the AdnB subunit ablate DNA-dependent ATPase activity, DSB end resection, and ATP-inducible ssDNA cleavage; the equivalent mutations of the AdnA subunit have comparatively little effect. AdnAB is a novel signature of the Actinomycetales taxon. Mycobacteria are exceptional in that they encode both AdnAB and RecBCD, suggesting the existence of alternative end-resecting motor–nuclease complexes.     

A new method for IgE detection in nasal mucosa

A new method for determining IgE antibodies on mucosal surfaces has been developed with the purpose of overcoming the main problems of nasal secretion sampling and standardization. The method is based on the principle of performing the incubation of the solid-phase coupled allergen with IgE antibody directly on the mucosal surface by means of a proper applicator. About two times higher values of specific IgE have been obtained with 5 min incubation on septal mucosa, behind the internal ostium, than with 3 hr in-vitro incubation with native secretion. In a study of 53 children with allergic rhinitis and asthma and 10 healthy non-atopic controls the sensitivity and specificity of this new method were evident. Because of its reliability and easy execution the new method could be widely used in diagnosis of allergic disease. Further, it seems to offer a good opportunity to study the IgE-mediated reaction in the target organ more extensively.     

A new method for measuring the pneumotachometer characteristics using a syringe

A new method for determining the nonlinear characteristics of pneumotachometers was developed. This method consists of a computer algorithm analyzing unsteady flows generated by a syringe. We removed the possible influence of the frequency characteristics of the measuring system by inserting an acoustic low pass filter in the path of gas flow. Two types of Fleisch pneumotachometers were tested. For a given volume of 3 l, the error of measured volume is 12.5% in uncorrected series and 0.2% in the corrected.     

汉族人群血管紧张素原基因的新单核苷酸多态性位点

应用改进的PCR－SSCP技术、多种聚丙烯酰胺凝胶电泳和PCR产物直接测序等方法，分析人的血管紧张素原（AGT）基因启动子区5远侧端的单核苷酸多态性（SNP）。在非变性胶上，发现不同样品的PCR产物（双链DNA分子）电泳迁移率不同；而在变性胶上，对应的单链DNA分子电泳迁移率相同；PCR产物直接测序的结果表明，在同一PCR扩增片段中同时存在两个SNPs位点（G－1074T，A－1179G）；通过476个不同样品的实验结果，进一步证实两个SNPs位点处于完全的连锁不平衡关系。GenBank同源性比较结果显示：A－1179G为中国汉族人群所特有。     

单管双向等位基因专一性扩增的单核苷酸多态分型的新方法

目的：建立一种基于等位基因专一性PCR原理的单核苷酸多态（single nucleotidepolymorphism,SNP)分型新方法：单管双向等位基因专一性扩增（single-tube bi-directional allele specificamplification,SB－ASA）,并考察专一性引物的3’端第3位碱基不配对对特异延伸的影响。方法：一个PCR反应体系包含两个3’末端分别与SNP两个等位基因特异结合的引物,它们延伸方向相反,产生长度不同的等位基因专一扩增产物,同时在两个等位基因特异性引物的3’端第3位碱基引入不配对以增加特异性。PCR产物经琼脂糖凝胶电泳后分析确定样本的基因型。观察在不同的温度条件下,近3’末端引入与不引入碱基不配对时两种引物特异延伸的情况,比较两种引物能特异延伸的退火温度（annealingtemperature,Ta)范围。结果：对于4个不同类型的SNP位点,SB－ASA都成功地分型了36个样本,与直接测序的结果完全一致。两条专一性引物3’物第3位碱基引入不配对后,能特异延伸的退火温度Ta范围分别从64℃～69℃、60℃～62℃扩大到46℃～66℃、56℃～61℃。结论：SB－ASA是一种简单快速而有效的SNP分型新方法;在等位基因专一性PCR体系中,专一性引物3’端第3位碱基引入不配对能增加引物对两个等位基因的区分能力。     

A novel method for isolation of neutrophils from murine blood using negative immunomagnetic separation

Inappropriate neutrophil activation has been implicated in the pathology of several clinically important inflammatory conditions. Although murine models are extensively used in the investigation of such pathological processes, a reliable method by which viable, quiescent neutrophils can be isolated from murine blood has not been developed. Here we describe a novel method based on negative immunomagnetic separation, which yields highly pure populations of murine neutrophils. Blood is incubated with a cocktail of antibodies against specific cell markers on unwanted cells, and then with secondary antibody-coated magnetic beads. After running the preparation through a column within a magnetic field, labeled cells are retained, and a neutrophil-rich effluent is collected. This method yields a >95% pure suspension of >97% viable neutrophils, recovering approximately 70% of neutrophils from whole blood. Flow cytometric analysis shows little difference in surface L-selectin and CD18 expression on isolated neutrophils compared with neutrophils in whole blood, indicating that neutrophils are minimally activated bythe isolation process. Stimulation with phorbol 12-myristate 13-acetate (PMA) reduced L-selectin andincreased CD18 expression. Isolated neutrophilsmigrate under agarose in response to fMLP, and fluorescently labeled neutrophils transfused into recipient mice interact with postcapillary venules in a manner comparable to endogenous leukocytes. These findings show that neutrophils isolated using this method can be used for inflammatory studies in vitro and in vivo.     

Development of a new molecular detection method for Taylorella equigenitalis

On PCR amplification of the intervening sequences (IVSs) in the central (helix 45) region within 23S rRNA gene sequences with T. equigenitalis (n = 34), as well as T. asinigenitalis (n = 35) and Bordetella (n = 11) isolates by using the primer pair of f-/r-23STis2, approximately 0.8 kb of the amplicons were generated, sequenced and analyzed. One IVS of approximately 70 bp in length was identified in all the Taylorella organisms but not Bordetella. PCR amplification was further developed for the convenient and rapid molecular detection of T. equigenitalis organisms with the IVS in the helix 45 region within the 23S rRNA genes as target by using the primer pairs (f-IVSde/r-23de). Thus, these results clearly demonstrated that PCR amplification with the primer pair (f-IVSde/r-23de) can be reliable in order to differentiate the T. equigenitalis isolates from both the T. asinigenitalis and Bordetella organisms.     

A strategy for detection of known and unknown SNP using a minimum number of oligonucleotides applicable in the clinical settings

Detection of unknown single nucleotide polymorphism (SNP) relies on large scale sequencing expeditions of genomic fragments or complex high-throughput chip technology. We describe a simplified strategy for fluorimetric detection of known and unknown SNP by proportional hybridization to oligonucleotide arrays based on optimization of the established principle of signal loss or gain that requires a drastically reduced number of matched or mismatched probes. The array consists of two sets of 18-mer oligonucleotide probes. One set includes overlapping oligos with 4-nucleotide tiling representing an arbitrarily selected "consensus" sequence (consensus-oligos), the other includes oligos specific for known SNP within the same genomic region (variant-oligos). Fluorescence-labeled DNA amplified from a homozygous source identical to the consensus represents the reference target and is co-hybridized with a differentially-labeled test sample. Lack of hybridization of the test sample to consensus- with simultaneous hybridization to variant-oligos designates a known allele. Lack of hybridization to consensus- and variant-oligos indicates a new allele. Detection of unknown variants in heterozygous samples depends upon fluorimetric analysis of signal intensity based on the principle that homozygous samples generate twice the amount of signal. This method can identify unknown SNP in heterozygous conditions with a sensitivity of 82% and specificity of 90%. This strategy should dramatically increase the efficiency of SNP detection throughout the human genome and will decrease the cost and complexity of applying genomic wide analysis in the context of clinical trials.     

A highly sensitive detection method for immunohistochemistry using biotinylated tyramine

A highly sensitive method for light microscopic immunohistochemistry is described. The increased sensitivity compared with current methodologies is based on the horseradish peroxidase-catalysed deposition of biotinylated tyramine at the sites of immunoreactivity, followed by the detection of the biotin with streptavidin biotin horseradish peroxidase complex. This method is of general applicability in immunohistochemistry and has several important advantages over currently used immunohistochemical detection procedures. The most significant advantage is that several antibodies which to date have been non-reactive, even in antigen-retrieval formalin-fixed, wax-embedded sections, now show strong and reproducible immunoreactivity using biotinylated tyramine amplification. In addition, many other antibodies can be used at significantly higher dilutions. © 1997 John Wiley & Sons, Ltd.