Multidirectional analysis for a colchicine poisoning case revealed detail cause of death and its mechanism.

The appearance of Meadow saffron (Colchicum autumnale), which contains colchicine, closely resembles Alpine leek (Allium victorialis), a popular edible wild vegetable in Northern Japan. This often results in the accidental ingestion of Meadow saffron and acute colchicine poisoning deaths. Here, we report on a case of acute colchicine poisoning death caused by the accidental ingestion of Meadow saffron. A man in his 70 s had been given wild vegetables from his neighborhood, which were then cooked and eaten by himself and his wife. Several hours later, they suffered from abdominal pain, vomiting, and diarrhea. They immediately went to the hospital and received routine treatment. While his wife made a full recovery, he died at home two days after consumption of the vegetables. A forensic autopsy was conducted five days after ingestion of the Meadow saffron and a lethal concentration (21.5 ng/mL) of colchicine in the peripheral blood sample was detected by liquid chromatography-tandem mass spectrometry. Distribution of colchicine in body fluids, tissues and gastrointestinal contents was also investigated. Some of the plants he had eaten were identified as Alpine leek or Meadow saffron by genetic analysis of his stomach contents. Histopathological examination showed apoptotic cells and cell cycle arrest at the metaphase in the intestinal crypts and testis. In addition, we detected high concentrations of endotoxins and tumor necrosis factor-α in his blood, indicating that intestinal mucosal injury induced by colchicine poisoning had allowed endotoxins to invade the body, causing death by endotoxin shock.

Acute Colchicine Poisoning Causes Endotoxemia via the Destruction of Intestinal Barrier Function: The Curative Effect of Endotoxin Prevention in a Murine Model.

BACKGROUND: Colchicine binds to intracellular tubulin and prevents mitosis. Colchicine is also used as an anti-inflammatory drug. Meanwhile, excess administration of medication or accidental ingestion of colchicine-containing plants can cause acute colchicine poisoning, which initially results in gastrointestinal effects that may be followed by multiorgan dysfunction. However, the mechanism of colchicine poisoning remains unclear, and there are no standard therapeutic strategies. AIMS: We focused on intestinal barrier function and attempted to reveal the underlying mechanism of colchicine poisoning using an animal model. METHODS: Colchicine was orally administered to C57Bl/6 mice. Then, we performed histopathological analysis, serum endotoxin assays, and intestinal permeability testing. Additionally, the LPS-TLR4 signaling inhibitor TAK-242 was intraperitoneally injected after colchicine administration to analyze the therapeutic effect. RESULTS: We observed villus height reduction and increased numbers of apoptotic cells in the gastrointestinal epithelium of colchicine-treated mice. Both intestinal permeability and serum endotoxin levels were higher in colchicine-treated mice than in control mice. Although colchicine-poisoned mice died within 25 h, those that also received TAK-242 treatment survived for more than 48 h. CONCLUSION: Colchicine disrupted intestinal barrier function and caused endotoxin shock. Therapeutic inhibition of LPS-TLR4 signaling might be beneficial for treating acute colchicine poisoning.

Hypothermia-induced activation of the splenic platelet pool as a risk factor for thrombotic disease in a mouse model.

BACKGROUND: Hypothermia, either therapeutically induced or accidental (ie, an involuntary decrease in core body temperature to <35°C), results in hemostatic disorders. However, it remains unclear whether hypothermia enhances or inhibits coagulation, especially in severe hypothermia. The present study evaluated the thrombocytic and hemostatic changes in hypothermic mice. METHODS: C57Bl/6 mice were placed at an ambient temperature of -20°C under general anesthesia. When the rectal temperature decreased to 15°C, 10 mice were immediately euthanized, while another 10 mice were rewarmed, kept in normal conditions for 24 hours, and then euthanized. These treatments were also performed in 20 splenectomized mice. RESULTS: The hypothermic mice had adhesion of CD62P-positive platelets with high expression of von Willebrand factor (vWF) in their spleens, while the status of the peripheral platelets was unchanged. Furthermore, the plasma levels of platelet factor 4 (PF4) and pro-platelet basic protein (PPBP), which are biomarkers for platelet degranulation, were significantly higher in hypothermic mice than in control mice, indicating that hypothermia activated the platelets in the splenic pool. Thus, we analyzed these biomarkers in asplenic mice. There was no increase in either PF4 or PPBP in splenectomized hypothermic mice. Additionally, the plasma D-dimer elevation and microthrombosis were caused in rewarmed mice, but not in asplenic rewarmed mice. CONCLUSIONS: Our results indicate that hypothermia leads to platelet activation in the spleen via the upregulation of vWF, and this activation causes hypercoagulability after rewarming.

Discrimination of haplotype in mitochondrial DNA mixtures using LNA-mediated PCR clamping.

Locked nucleic acid (LNA) has been widely used for various genetic analyses, and has many benefits, in terms of the specificity or sensitivity of amplification, because LNA-containing primers/probes form more stable duplexes with template DNA than probes lacking LNA. Here, we developed a new method for discriminating HV1 haplotypes from mitochondrial DNA (mtDNA) mixtures by applying PCR clamping using LNA. PCR clamping is based on the selective inhibition of amplification using LNA-containing probes, which can discriminate single-nucleotide differences. Before designing probes, we selected 171 sequences with single-nucleotide variations from the HV1 region, and evaluated the specificity of LNA-containing probes for them by predicting Tm values. The differences of Tm between mismatched and exactly matched probe-template duplexes depended markedly on the type of LNA nucleotides for discriminating single-nucleotide differences, and the cytosine LNA nucleotide at the site of variations in the probes was most effective to discriminate these differences. For mixture analysis, each probe targeted one or two variations (16209C, 16217C, 16257A/16261T, 16297C/16298C, 16304C, 16362C, or 16362T) that are particularly common in the Japanese population, and seven designed probes completely inhibited the amplification of exactly matched templates. We prepared mixed samples by mixing DNA from two individuals at a ratio of 1:9, 1:4, 1:1, 4:1, or 9:1, and then performed Sanger sequencing analysis after PCR clamping with each probe. Our method distinguished each haplotype at lower ratios from two-person mixtures, and enabled sensitive detection at 12 pg of total DNA including 600 copies of mtDNA. Moreover, we analyzed three-person mixtures with representative sequences, and detected the minor haplotype of one individual present at a rate of 10% by adding two selected probes. The ability to discriminate haplotypes in mixed samples by using LNA-mediated PCR clamping indicates the potential value of mtDNA analysis in criminal investigations.

Assessment of DNA degradation of buccal cells under humid conditions and DNA repair by DOP-PCR using locked nucleic acids.

We analyzed the degradation level of DNA from buccal cells under humid conditions using quantitative PCR analysis. Gauze samples with buccal cells were incubated for up to 12 months under three different conditions (25 °C/dry, 25 °C/humid, or 40 °C/humid). The degradation was evaluated based on two degradation ratios (129:41 and 305:41 bp). DNA degraded slowly under the 25 °C/humid condition, and significant differences in the two degradation ratios were detected between 25 °C/dry and 25 °C/humid conditions after 12 months. Moreover, the degradation rapidly progressed under the 40 °C/humid condition, and the two degradation ratios in this condition were much lower than those from 25 °C/dry and 25 °C/humid conditions after a short incubation period (3 months). To evaluate the effect of DNA repair on low-copy degraded DNA, degenerate oligonucleotide-primed PCR (DOP-PCR) was performed before short tandem repeats (STR) genotyping. As a standard DOP-PCR, we used a 22-base primer with 10 degenerate sequences (5'-CTCGAGNNNNNNNNNNATGTGG-3'), and additionally designed DOP-PCR primers with 2, 4, 6, or 8 locked nucleic acids (LNAs). When slightly degraded DNA (305:41-bp ratio = 0.60) was used, DOP-PCR significantly increased the fluorescent intensity and success rate of genotyping using Identifiler and Globalfiler kits. In particular, the reaction with four LNAs produced the highest value. However, such benefits were not observed in the analysis of moderately degraded DNA (305:41-bp ratio = 0.13). Although the recovery rates of STR profiles by DOP-PCR were dependent on the degradation level of low-copy DNA, the effectiveness of DOP-PCR highlights the potential of LNA for degenerate sequences.

Paraquat toxicity is attenuated by 4-phenylbutyrate-induced phosphorylation of ERK2 via PI3K in A549 cells.

Paraquat (PQ) is a widely used herbicide in the world despite being highly toxic to humans. PQ causes fatal damage to multiple organs, especially the lungs. While oxidative stress is the main toxic mechanism of PQ, there is no established standard therapy for PQ poisoning. In this study, we investigated the cytoprotective effect of 4-phenylbutyrate (4PBA) on PQ toxicity in human lung adenocarcinoma A549 cells. Phosphorylation levels of major survival signaling kinases Akt and ERK, as well as expression levels of antioxidant enzymes catalase and superoxide dismutase 2 (SOD2) were examined. The cytoprotective mechanism of 4PBA against PQ was compared with the antioxidant reagent trolox. We demonstrated that both 4PBA and trolox attenuated PQ toxicity, but their mechanisms were different. 4PBA increased ERK2 phosphorylation levels, which could be inhibited by the PI3K inhibitor LY294002. The cytoprotective effect of 4PBA was also inhibited by LY294002. Catalase expression levels were increased by 4PBA, although this increase was not inhibited by LY294002. 4PBA did not increase SOD2 expression. Trolox did not affect phosphorylation of Akt or ERK, or the expression of antioxidant enzymes. These results suggest that 4PBA attenuated PQ cytotoxicity by ERK2 activation via PI3K. Our study may provide new findings for understanding the molecular mechanism underlying cytoprotection by 4PBA, as well as new therapeutic targets for PQ poisoning.

Liquid chromatography-tandem mass spectrometry method for the determination of thiosulfate in human blood and urine as an indicator of hydrogen sulfide poisoning.

Being a stable metabolite of hydrogen sulfide, thiosulfate has been utilized as an index for hydrogen sulfide poisoning (HSP). Thiosulfate analysis is mainly performed using gas chromatography/mass spectrometry (GC-MS) due to its high sensitivity and specificity. The GC-MS analysis requires two-step derivatizations of thiosulfate, and the derivative is not stable in solution as it has a disulfide moiety. To resolve this stability issue, we developed a novel analytical method using liquid chromatography-tandem mass spectrometry (LC-MS/MS) for monitoring the pentafluorobenzyl derivative of thiosulfate (the first reaction product of the GC-MS method) in this study. The established method exhibited high reproducibility despite being a more simplified and rapid procedure compare to the GC-MS method. Phenyl 4-hydroxybenzoate was used as an internal standard because 1,3,5-tribromobenzene which had been used in the GC-MS method was not suitable compound for LC-MS/MS with Electrospray ionization (ESI) negative detection. The linear regression of the peak area ratios versus concentrations was fitted over the concentration ranges of 0.5-250µM and 0.25-250µM in blood and urine, respectively. The validation results satisfied the acceptance criteria for intra- and inter-day accuracy and precision. Blood and urine samples from 12 suspected HSP cases were tested using this method. The thiosulfate concentration detected in the sample coincided well with that determined at the scene of each HSP accident.

Postmortem diffusion of n-butane and i-butane used for anticontagious plugging spray.

Blood and tissue samples from a forensic autopsy of a man in his late 60s, who developed dementia and died of multiple head traumas due to a fall from a moving vehicle, contained certain amounts of n-butane and i-butane. The concentration of n-butane was in the range of 0.48-70.5 µL/g, which would be considered as toxic or lethal levels. We had to distinguish whether the cause of his unexplained behavior was due to his pre-existing condition (dementia), or from a confused state induced by butane abuse. No traces of butane use were found at the scene. Police investigation revealed that a propellant used in an anticontagious plugging spray had been administered to him during a postmortem treatment in the emergency hospital. In order to prove the postmortem butane diffusion had resulted from the spray administration and to estimate the diffused concentration, experimental simulation was conducted by using rats. As a result of postmortem treatment with the spray, n-butane at concentrations of 0.54-15.5 µL/mL or g were found in the rat blood and tissues. In this case, we provided further evidence that the postmortem butane diffusion, caused by using the anticontagious plugging spray containing butane gas as a propellant administered to a cadaver during a postmortem procedure prior to forensic autopsy, should be distinguished from cases of actual butane poisoning.

A Case of Sudden Infant Death Due to Incomplete Kawasaki Disease.

Although Kawasaki disease (KD) is a self-limiting disease, it may cause sudden cardiac death. Diagnosis of KD is principally based on clinical signs; however, some infant cases do not meet the criteria. Such cases are identified as incomplete KD. The sudden death risk in incomplete KD cases is similar to conventional KD. In our 5-month-old case, he had been admitted to a hospital for a fever and suppuration at the site of Bacille de Calmette et Guerin (BCG) vaccination. However, after discharge from the hospital, his C-reactive protein (CRP) levels declined, he got indisposed and died suddenly. A medico-legal autopsy revealed myocarditis, coronaritis, platelet-aggregated emboli in coronary arteries, and myocardial degeneration, suggesting that the fatal myocardial infarction was due to thrombus emboli in the coronary arteries. Forensic pathologists therefore should pay attention to the cardiac pathology originated from incomplete KD as a potential cause in cases of sudden infant death.

Multicolor-based discrimination of 21 short tandem repeats and amelogenin using four fluorescent universal primers.

The aim of this study was to develop a cost-effective genotyping method using high-quality DNA for human identification. A total of 21 short tandem repeats (STRs) and amelogenin were selected, and fluorescent fragments at 22 loci were simultaneously amplified in a single-tube reaction using locus-specific primers with 24-base universal tails and four fluorescent universal primers. Several nucleotide substitutions in universal tails and fluorescent universal primers enabled the detection of specific fluorescent fragments from the 22 loci. Multiplex polymerase chain reaction (PCR) produced intense FAM-, VIC-, NED-, and PET-labeled fragments ranging from 90 to 400 bp, and these fragments were discriminated using standard capillary electrophoretic analysis. The selected 22 loci were also analyzed using two commercial kits (the AmpFLSTR Identifiler Kit and the PowerPlex ESX 17 System), and results for two loci (D19S433 and D16S539) were discordant between these kits due to mutations at the primer binding sites. All genotypes from the 100 samples were determined using 2.5 ng of DNA by our method, and the expected alleles were completely recovered. Multiplex 22-locus genotyping using four fluorescent universal primers effectively reduces the costs to less than 20% of genotyping using commercial kits, and our method would be useful to detect silent alleles from commercial kit analysis.

An objective approach using three indexes for determining fatal hypothermia due to cold exposure; statistical analysis of oxyhemoglobin saturation data.

Analysis of oxyhemoglobin (O2-Hb) saturation levels in the left and right heart blood is useful in the assessment of exposure to cold surroundings before death. We quantified conventional subjective visual evaluation of O2-Hb saturation levels and developed useful diagnostic criteria for fatal hypothermia: O2-Hb saturation in the left heart blood (L-O2Hb) was ⩾36%, the O2-Hb saturation gap between the left and right heart blood (L-R gap) was ⩾13%, and the O2-Hb saturation ratio of the left to right heart blood (L/R ratio) was ⩾1.8. When we used L-O2Hb of ⩾36% as a basic criterion and applied a further criterion of an L-R gap of ⩾13% or an L/R ratio of ⩾1.8, these criteria registered a sensitivity level of ⩾86% and specificity level of ⩾93% for the diagnosis of fatal hypothermia. This method can be useful for determining fatal hypothermia in connection with conventional autopsy findings, as well as histological and biochemical markers.

Rapid genotyping of 25 autosomal STRs in a Japanese population using fluorescent universal primers containing locked nucleic acids.

Amplification of fluorescently labeled products is one of the most popular methods for genotyping genetic variations. Two-step amplification using fluorescent universal primers simultaneously produces multiple targeted fragments labeled with fluorescent dyes, and this strategy is applicable to large-scale, cost-effective genotyping. In this study, we developed a fast PCR-based, multiple short tandem repeat (STR) genotyping method using fluorescent universal primers containing locked nucleic acids (LNAs). Four amplification reactions, each assaying six or seven markers and using 0.5-1.0 ng of genomic DNA, produced obvious Fam-labeled peaks in all 26 loci tested (25 autosomal STRs and amelogenin). The overall amplification time was 37 min. Moreover, fluorescent signals for the 25 STRs obtained from LNA-containing primers were 1.5-9.0 fold higher compared to those from non-LNA primers. Using genomic DNA from 120 Japanese individuals, 16 out of the 25 STRs had observed heterozygosity greater than 0.7. Some of these 25 STRs also had high discriminatory power, similar to that of the 13 core STRs in the Combined DNA Index System dataset. The probability of incorrectly assigning a match based on the accumulated matching probability for these 25 STRs is 1.2 × 10(-22), and their combined use can provide robust information for Japanese forensics.

Brain stem hemorrhage due to cerebral amyloid angiopathy: the autopsy of a patient with Alzheimer's disease at a young age.

We report findings from an autopsy of a male in his 40s who died of a brain stem hemorrhage associated with cerebral amyloid angiopathy (CAA), senile plaques (SPs) and neurofibrillary tangles (NFTs), which are histopathological changes associated with Alzheimer's disease (AD). Our immunohistochemical study demonstrated amyloid ß (Aß) deposition in the small cerebral arteries and SPs. Although hypertension (178/132 mmHg) was detected, the subject was not treated accordingly. CAA coupled with hypertension might have caused the intracerebral hemorrhage (ICH).

Genotyping of 38 insertion/deletion polymorphisms for human identification using universal fluorescent PCR.

Short insertion/deletion (Indel) polymorphisms of approximately 2-6 bp are useful as biallelic markers for forensic analysis, and the application of Indel genotyping as a supplementary tool would improve human identification accuracy. We examined the allele frequencies of 37 autosomal Indels in the Japanese population and developed a novel dual-color genotyping method for human identification on the basis of universal fluorescent PCR, including the sex-typing amelogenin locus. Target genomic fragment sizes for 38 Indels were 49-143 bp. We analyzed these Indels in 100 Japanese individuals using the M13(-47) sequence as a universal primer. For dual-color genotyping, we designed a novel universal primer with high amplification efficiency and specificity. Using FAM-labeled M13(-47) and HEX-labeled modified M13(-47) primers, fluorescent signals at all loci were clearly distinguished in two independent multiplex PCRs. Average minor allele frequency was 0.39, and accumulated matching probability was 2.12 × 10(-15). Complete profiles were successfully amplified with as little as 0.25 ng of DNA. This method provides robust, sensitive, and cost-effective genotyping for human identification.

Sodium tauroursodeoxycholate prevents paraquat-induced cell death by suppressing endoplasmic reticulum stress responses in human lung epithelial A549 cells.

Paraquat is a commonly used herbicide; however, it is highly toxic to humans and animals. Exposure to paraquat causes severe lung damage, leading to pulmonary fibrosis. However, it has not been well clarified as how paraquat causes cellular damage, and there is no established standard therapy for paraquat poisoning. Meanwhile, endoplasmic reticulum stress (ERS) is reported to be one of the causative factors in many diseases, although mammalian cells have a defense mechanism against ERS-induced apoptosis (unfolded protein response). Here, we demonstrated that paraquat changed the expression levels of unfolded protein response-related molecules, resulting in ERS-related cell death in human lung epithelial A549 cells. Moreover, treatment with sodium tauroursodeoxycholate (TUDCA), a chemical chaperone, crucially rescued cells from death caused by exposure to paraquat. These results indicate that paraquat toxicity may be associated with ERS-related molecules/events. Through chemical chaperone activity, treatment with TUDCA reduced paraquat-induced ERS and mildly suppressed cell death. Our findings also suggest that TUDCA treatment represses the onset of pulmonary fibrosis caused by paraquat, and therefore chemical chaperones may have novel therapeutic potential for the treatment of paraquat poisoning.

Universal fluorescent labeling of amplification products using locked nucleic acids.

Amplification/hybridization-based genetic analyses using primers containing locked nucleic acids (LNAs) present many benefits. Here, we developed a novel design for universal fluorescent PCR using LNAs. Universal fluorescent PCR generates intermediate nonlabeled fragments and final fluorescent fragments in a two-step amplification process that uses locus-specific primers with universal tails and universal fluorescent primers. In this study, a few standard nucleotides were replaced with LNAs only in the fluorescent universal primers. The sequence of the fluorescent universal primer significantly affected the amplification efficiency. For primers with three LNAs, the fluorescent primers with stable M13(-47) sequences provided the most efficient signal (approximately tenfold higher than the primers with M13(-21) sequences at lower Tm values). Moreover, AT-rich LNA substitutions in the fluorescent primers produced much lower amplification efficiencies than GC-rich substitutions. GC-rich LNAs produced greater differences in Tm values among primers, and resulted in the preferential production of fluorescently labeled amplicons. The specificity and sensitivity of LNA-containing fluorescent primers were assessed by genotyping eight STRs in Japanese individuals, and full STR profiles could be generated using as little as 0.25 ng of genomic DNA. The method permitted clear discrimination of alleles and represents sensitive STR genotyping at a reduced cost.

Estimation of the duration after methamphetamine injection using a pharmacokinetic model in suspects who caused fatal traffic accidents.

When the population parameters of drug pharmacokinetics in the human body system are known, the time-course of a certain drug in an individual can generally be estimated by pharmacokinetics. In the present two cases where methamphetamine abusers were suspected to have inflicted mortalities in traffic accidents, the time-elapse or duration immediately after methamphetamine injection to the time when the accidents occurred became points of contention. In each case, we estimated the time-course of blood methamphetamine after the self-administration in the suspects using a 2-compartment pharmacokinetic model with known pharmacokinetic parameters from the literatures. If the injected amount can be determined to a certain extent, it is easy to calculate the average time-elapse after injection by referring to reference values. However, there is considerable individual variability in the elimination rate based on genetic polymorphism and a considerably large error range in the estimated time-elapse results. To minimize estimation errors in such cases, we also analyzed genotype of CYP2D6, which influenced methamphetamine metabolism. Estimation based on two time-point blood samples would usefully benefit legal authorities in passing ruling sentences in cases involving similar personalities and circumstances as those involved in the present study.

Multiplex PCR-based Alu insertion polymorphisms genotyping for identifying individuals of Japanese ethnicity.

Discrimination of Alu insertions is a useful tool for geographic ancestry analysis, and is usually performed by Alu element amplification and agarose gel electrophoresis. Here, we have developed a new fluorescence-based method for multiple Alu genotyping in forensic identification. Allele frequencies were determined in 70 Japanese individuals, and we selected 30 polymorphic Alu insertions. Three primers were designed for each Alu locus to discriminate alleles using the 3-6 bp differences in amplicon sizes. Furthermore, we classified the amplification primers for the 30 loci into three different sets, and PCR using each set of primers provided 10 loci fragments ranging from 50 to 137 bp. Based on population data, the probability of incorrectly assigning a match was 3.7×10(-13). Three independent amplifications and subsequent capillary electrophoresis enabled the sensitive genotyping of small amounts of DNA, indicating that this method is suitable for identifying individuals of Japanese ethnicity.

HRD1 levels increased by zonisamide prevented cell death and caspase-3 activation caused by endoplasmic reticulum stress in SH-SY5Y cells.

Zonisamide, which is commonly prescribed at high doses (200-400 mg/day) for the treatment of partial seizures, has recently been used at a low dose (25 mg/day) for improving parkinsonian syndrome. However, the molecular mechanisms that underlie the antiparkinsonian effects of zonisamide have not been clarified. Here we show that low micromolar concentrations of zonisamide prevented cleavage of caspase-3 and cell death in human dopaminergic SH-SY5Y neuroblastoma cells that were subjected to endoplasmic reticulum stress induced by tunicamycin or 6-hydroxydopamine. Hypodense zonisamide increased the expression levels of SEL1L, which is known to stabilize the ubiquitin ligase HRD1. Indeed, upregulation of HRD1 protein was observed. Thus, the results of this study strongly suggest that low concentrations of zonisamide inhibit neuronal cell death by increasing HRD1 protein levels in patients with Parkinson's disease. Consequently, in addition to the treatment of Parkinson's disease, the therapeutic potential of zonisamide should be considered for the treatment of several neurodegenerative disorders with pathophysiological mechanisms involving endoplasmic reticulum stress.

Enhanced discrimination of single nucleotide polymorphisms using 3' nucleotide differences in ligase detection reaction probes.

The ligase detection reaction (LDR) is a highly specific genotyping method for single nucleotide variations. Although LDR typically discriminates single nucleotide polymorphism (SNP) alleles at the 3' end of so-called LDR discriminating probes, we designed probes in which the position of nucleotide differences for discrimination was shifted to the second and third nucleotides from the 3' end. Using the 3'-modified probes, we targeted SNPs of the human ABO group and investigated the specificity and efficiency of ligation by a universal LDR assay. We demonstrated that one or two nucleotide shifts of differences in discriminating probes improve the allele balance in detecting both base substitutions and short deletions. In regard to short deletions, moreover, the shifts of nucleotide differences in discriminating probes form the perfect-machted or multiple-mismatched structures (the bulge structures) in the discriminating probe-target DNA duplex and may contribute to enhance ligation efficiency.