Simultaneous disturbance of NHE1 and LOXL2 decreases tumorigenicity of head and neck squamous cell carcinoma.

OBJECTIVE: Although there have been brilliant advancements in the practical application of therapies targeting immune checkpoints, achieving success in targeting the microenvironment remains elusive. In this study, we aimed to address this gap by focusing on Na+ / H+ exchanger 1 (NHE1) and Lysyl Oxidase Like 2 (LOXL2), which are upregulated in head and neck squamous cell carcinoma (HNSCC) cells. METHODS: The malignancy of a metastatic human HNSCC cell line was assessed in a mouse tongue cancer xenograft model by knocking down (KD) NHE1, responsible for regulating intracellular pH, and LOXL2, responsible for extracellular matrix (ECM) reorganization via cross-linking of ECM proteins. In addition to assessing changes in PD-L1 levels and collagen accumulation following knockdown, the functional status of the PD-L1 / PD-1 immune checkpoint was examined through co-culture with NK92MI, a PD-1 positive phagocytic human Natural Killer (NK) cell line. RESULTS: The tumorigenic potential of each single KD cell line was similar to that of the control cells, whereas the potential was attenuated in cells with simultaneous KD of both factors (double knockdown [dKD]). Additionally, we observed decreased PD-L1 levels in NHE1 KD cells and compromised collagen accumulation in LOXL2 KD and dKD cells. NK92MI cells exhibited phagocytic activity toward HNSCC cells in co-culture, and the number of remaining dKD cells after co-culture was the lowest in comparison to the control and single KD cells. CONCLUSION: This study demonstrated the possibility of achieving efficient anti-tumor effects by simultaneously disturbing multiple factors involved in the modification of the tumor microenvironment.

The anti-inflammatory and anti-oxidative effect of a classical hypnotic bromovalerylurea mediated by the activation of NRF2.

The Kelch-like ECH-associated protein 1-nuclear factor erythroid 2-related factor 2 (KEAP1-NRF2) system plays a central role in redox homeostasis and inflammation control. Oxidative stress or electrophilic compounds promote NRF2 stabilization and transcriptional activity by negatively regulating its inhibitor, KEAP1. We have previously reported that bromovalerylurea (BU), originally developed as a hypnotic, exerts anti-inflammatory effects in various inflammatory disease models. However, the molecular mechanism underlying its effect remains uncertain. Herein, we found that by real-time multicolor luciferase assay using stable luciferase red3 (SLR3) and green-emitting emerald luciferase (ELuc), BU potentiates NRF2-dependent transcription in the human hepatoblastoma cell line HepG2 cells, which lasted for more than 60 h. Further analysis revealed that BU promotes NRF2 accumulation and the transcription of its downstream cytoprotective genes in the HepG2 and the murine microglial cell line BV2. Keap1 knockdown did not further enhance NRF2 activity, suggesting that BU upregulates NRF2 by targeting KEAP1. Knockdown of Nfe2l2 in BV2 cells diminished the suppressive effects of BU on the production of pro-inflammatory mediators, like nitric oxide (NO) and its synthase NOS2, indicating the involvement of NRF2 in the anti-inflammatory effects of BU. These data collectively suggest that BU could be repurposed as a novel NRF2 activator to control inflammation and oxidative stress.

Excised human larynx in N-vinyl-2-pyrrolidone-embalmed cadavers can produce voiced sound by pliable vocal fold vibration.

Tissue-hardening effect and health-hazard issue of formaldehyde (FA) have long been a great disadvantage of this conventional fixative in anatomical research. We recently developed a FA-free embalming method for cadavers which utilizes N-vinyl-2-pyrrolidone (NVP) and enables assessment of motion kinetics by maintaining the softness of embalmed tissue. By assessing the feasibility of NVP-embalmed tissue to mimic vocalization, this study aimed to prove the potential of embalmed cadavers, which have previously been used only for the understanding of anatomical morphology, for the assessment of precise motion physiology in the human body. Ten cadavers embalmed in NVP (n = 6) and FA (n = 4) were incorporated in this study. Excised larynges underwent experimental phonation to mimic vocalization with fast and pliable vibration of vocal folds. High-speed digital imaging was utilized for the assessment of vocal fold vibration. Furthermore, acoustic analysis of the voiced sound, and reproducibility examination were also performed. Regular vocal fold vibrations successfully produced voiced sounds during experimental phonation using NVP-embalmed larynges. The vibratory frequency, vibration amplitude, and stretch rate of the vocal folds were comparable to those of living humans. Six months after the first experiment, the vocal parameters were reproduced, to suggest the long-term preservation potential of our NVP-embalming technique. On the other hand, neither voiced sound nor vocal fold vibration were observed in FA-embalmed larynges. This novel embalming technique could pioneer the next era to utilize embalmed cadavers for the examination of motion physiology in the human body.

Ergonomic advantage of pistol-grip endoscope in the ENT practice.

OBJECTIVES: Recent technology manufactured a nasopharyngeal videoscope with pistol-shaped grip (PG). This study aimed to assess the ergonomic feasibility of this novel device in daily ENT practice. METHODS: To assess the ergonomic impact of grip shape on ENT physicians, conventional grip videoscope (CG) and PG were utilized in this study. Surface electromyography (sEMG) was recorded to assess the muscle activity in the upper limb during endoscopy on a training model. Bilateral sEMG recordings were performed including thenar muscle, pronator teres muscle, brachioradialis muscle, and biceps brachii muscle. Mean value of the mean sEMG amplitude throughout the task in triplicated examinations (mMA) with each electrode, total values of four mMAs in both of the grip-side and the insertion tube-side limb muscles, and total value of all eight mMAs were calculated, and compared between CG and PG. Subgroup analyses were also performed in the experienced ENT physicians and the residents. RESULTS: PG provided significantly lower mMA values in thenar muscle and brachioradialis muscle of the grip-side limb compared with CG. Total value of four mMAs in PG was significantly lower compared with that in CG in the grip-side limb, and total value of all eight mMAs in PG was significantly lower compared with that in CG. Furthermore, total value of four mMAs in PG was significantly lower compared with that in CG in the grip-side limb, in both of the subgroups. CONCLUSION: This is the first study to support the idea that the newly designed pistol-grip endoscope may have an ergonomic advantage over conventional endoscope for otolaryngologists in daily practice. LEVEL OF EVIDENCE: 4.

Three-dimensional imaging of vocalizing larynx by ultra-high-resolution computed tomography.

PURPOSE: Ultra-high-resolution computed tomography (UHRCT) is an emerging imaging technology that is able to achieve simultaneous 160 slices with super-thin 0.25 mm thickness. The purpose of this study was to assess the feasibility of UHRCT to visualize laryngeal structure and kinetics. METHODS: Three normal volunteers and three patients with unilateral vocal fold paralysis (UVFP) were incorporated in this case series. First, images were taken under five conditions in normal volunteers. Five tasks consisted of (1) air inspiration through the nose (IN), (2) breath holding (BH), (3) sustained vowel /i:/ phonation (IP), (4) humming phonation (HP), and (5) forced glottic closure during exhalation (FC). Three-dimensional CT images of arytenoid and cricoid cartilages, as well as virtual laryngoscopic images, were reconstructed using UHRCT data. Reconstructed images were compared among five conditions to assess the best tasks to picture laryngeal kinetics. Second, pre- and post-phonosurgical images were examined in UVFP patients to evaluate potential role of UHRCT to assess laryngeal pathology in hoarse patients. RESULTS: Among the five conditions, IN and IP conditions were considered suitable to visualize laryngeal structure at rest and during phonation, respectively. Kinetic abnormalities including asymmetric motion of arytenoid cartilages were elucidated in UVFP patients, and virtual endoscopy visualized the clinically invisible posterior three-dimensional glottic chinks. Furthermore, UHRCT was useful to understand changes in laryngeal structure achieved by phonosurgery. CONCLUSIONS: UHRCT is an emerging imaging technology that can be used for minimally invasive visualization and assessment of laryngeal structure and kinetics. Future studies to assess more number of patients with laryngeal dysfunction are warranted.

Pre-anesthetic Whole Body Point-of-care Ultrasonography.

Point-of-care ultrasonography has become widely used in diagnosis and managements of patients. In the field of anesthesiology, ultrasound guided nerve blocks and central venous catheterization (CVC), and transesophageal echocardiography (TEE) have become popular. Now, ultrasound devices are familiar to anesthesiologists. In pre-anesthetic patient risk evaluation, the sonographic results of transthoracic echocardiography (TTE) or the examination of deep venous thrombus are often helpful for anesthesia planning. Both examinations usually have been performed by sonographer. However, the ultrasonographic assessment of neck vessels, lung condition with diaphragm function are still less familiar among anesthesiologists. Here, we have introduced a series of pre-anesthetic whole body ultrasound evaluation of the patients. Because of less invasive technique, whole body ultrasound evaluation is suitable to use when anesthesiologists may have questions whether the patient's condition is good or not. No doubt that point-of-care ultrasonography by anesthesiologists themselves in pre-anesthesia. clinic can be the useful decision making tool of anesthesia plan. To be tomorrow's anesthesiologists, please start point-of-care pre-anesthetic ultrasonographic assessment.

[A Prospective Evaluation of the Fixation of the Catheters for Continuous Femoral Nerve Block in Patients Receiving Total Knee Arthroplasty].

BACKGROUND: Continuous femoral block in total knee arthroplasty gives excellent perioperative pain control and quality of rehabilitation. Although accidental catheter dislodgement and fluid leakage from the puncture site are considered as major complications, there is no standard catheter fixation technique introduced. We tested a novel technique using a new transparent medical dressing combined with instant tissue adhesive to reduce the catheter related complications. METHODS: Twenty patients were randomly divided into the Tegadarm comfort film dressing with aron alpha A [Sankyo] group (TA group) and the Tegadarm film group (T group). We measured the number of exchanged dressings, fluid leakage and catheter stability. RESULTS: TA group had significantly fewer number of exchanged dressings and fluid leakage than in the T group. The catheters were more stable in the TA group. There were no differences in adverse effects. CONCLUSIONS: Using the Tegadarm comfort film dressing with aron alpha A [Sankyo] could be an effective fixation method in the continuous femoral nerve block.

Alkyl hydroperoxide reductase enhances the growth of Leuconostoc mesenteroides lactic acid bacteria at low temperatures.

Lactic acid bacteria (LAB) can cause deterioration of food quality even at low temperatures. In this study, we investigated the cold-adaptation mechanism of a novel food spoilage LAB, Leuconostoc mesenteroides NH04 (NH04). L. mesenteroides was isolated from several spoiled cooked meat products at a high frequency in our factories. NH04 grew rapidly at low temperatures within the shelf-life period and resulted in heavy financial losses. NH04 grew more rapidly than related strains such as Leuconostoc mesenteroides NBRC3832 (NBRC3832) at 10°C. Proteome analysis of NH04 demonstrated that this strain produces a homolog of alkyl hydroperoxide reductase--AhpC--the expression of which can be induced at low temperatures. The expression level of AhpC in NH04 was approximately 6-fold higher than that in NBRC3832, which was grown under the same conditions. Although AhpC is known to have an anti-oxidative role in various bacteria by catalyzing the reduction of alkyl hydroperoxide and hydrogen peroxide, the involvement of AhpC in cold adaptation of food spoilage bacteria was unclear. We introduced an expression plasmid containing ahpC into NBRC3832, which grows slower than NH04 at 10°C, and found that expression of AhpC enhanced growth. These results demonstrated that AhpC, which likely increases anti-oxidative capacity of LAB, plays an important role in their rapid growth at low temperatures.

Association of inflammatory biomarkers with depressive symptoms and cognitive decline in a community-dwelling healthy older sample: a 3-year follow-up study.

BACKGROUND: The relationship between the pathophysiology of dementia and neuroinflammation is well-known. The number of reports stating that depression is a risk factor for dementia has recently been increasing. These epidemiological findings suggest the possibility that both depression and dementia have common pathophysiological backgrounds of neuroinflammation. METHODS: The sample consists of 64 non-demented community-dwelling older participants aged 65 years or over. Participants were assessed at baseline (2004-2006) and 3 years later (2007-2009). Plasma concentration of markers of inflammation (interleukins (IL)-1ß, IL-2, IL-6, soluble interleukin-2 receptor (sIL-2R), soluble interleukin-6 receptor (sIL-6R), high sensitivity C-reactive protein (hsCRP) and tumor necrosis factor (TNF)-α) were measured at baseline. Depression symptoms were assessed with the Beck Depression Inventory (BDI) and cognitive decline was assessed with the Mini-Mental State Examination (MMSE), Frontal Assessment Battery (FAB) and Clock Drawing Test (CDT) at baseline and follow-up. All analyses were adjusted for age, gender and years of education. RESULTS: In the cross-sectional analysis, the present study found soluble IL-2 receptor (sIL-2R) to be associated only with the MMSE score at baseline in men. In the longitudinal analysis, none of our inflammatory biomarkers were associated with either depressive symptoms or cognitive decline. LIMITATIONS: The present study consists of small number of participants and body mass index (BMI) scores were not obtained. CONCLUSIONS: Our findings suggest that sIL-2R is associated with current cognitive function in men. None of our inflammatory markers predicted future depressive state or cognitive decline in our community-dwelling healthy older sample.

The degree of N-glycosylation affects the trafficking and cell surface expression levels of Kv1.4 potassium channels.

Kv1.4 potassium channels are heavily glycosylated proteins involved in shaping action potentials and in neuronal excitability and plasticity. Kv1.4 N354Q, without an N-glycan, exhibited decreased protein stability and trafficking to the cell surface (Watanabe et al. in J Biol Chem 279:8879-8885, 2004). Here we investigated whether the composition of the N-glycan affected Kv1.4 cell surface expression. Kv1.4 proteins carrying N-glycans with different compositions were generated by adding glycosidase inhibitors or using N-glycosylation-deficient mutant cell lines. We found that oligomannose-type, hybrid-type, or incomplete complex-type N-glycans had a negative effect on surface protein expression of Kv1.4 compared with complex-type N-glycans. The decrease in surface protein level of Kv1.4 was mainly due to a reduction in total protein level, induced by altered N-glycan composition. Kv1.4 in CSTP-treated cells carried a unique oligomannose-type N-glycan that contains three glucose residues. This N-glycan had the most negative effect on cell surface expression of Kv1.4. It decreased Kv1.4 surface protein level by a combined mechanism of reducing total protein level and increasing ER-retention. Our data suggest that composition of the N-glycan plays an important role in protein stability and trafficking, and a sialylated complex-type N-glycan promoted high cell surface expression of Kv1.4.

Belief in life after death, salivary 3-methoxy-4-hydroxyphenylglycol, and well-being among older people without cognitive impairment dwelling in rural Japan.

OBJECTIVES: Research has found that spirituality/religiosity has a salutary association with mental/physical health. However, the association of belief in life after death with well-being has rarely been studied, and the same is true of its association with biological indices, such as monoamine transmitters. Therefore, we examined the associations between well-being and religiosity, salivary 3-methoxy-4-hydroxyphenylglycol (sMHPG), and demographic characteristics. METHODS: The participants were 346 community-dwelling people, aged 65 years or older, without cognitive or mental deficits, in rural Japan. Measures of religiosity consisted of belief in life after death, attachment to life, and experiences related to death and religion. The measures were assessed by scales specifically suited for Japanese religious orientations. Participants' well-being was assessed by a life satisfaction scale containing two subscales. We also measured sMHPG, a major metabolite of noradrenaline that is thought to reflect certain psychological states, such as psychomotor retardation and effortful attention. RESULTS: One subscale of life satisfaction was positively associated with belief in life after death and sMHPG, and the other life satisfaction subscale was positively associated with education and death/religion-related experiences (e.g., visiting family graves or loss of a friend). Gender differences were found in afterlife beliefs and each life satisfaction subscale. CONCLUSIONS: These results suggest that religiosity, including belief in life after death and death/religion-related experiences, is salubriously associated with mental health among older people, especially women, living in rural Japan. The basal level of sMHPG was positively associated with life satisfaction, but not with belief in life after death.

Role of VPAC2 receptor in monocrotaline-induced pulmonary hypertension in rats.

Pulmonary hypertension (PH) is associated with significant morbidity and mortality. Vasoactive intestinal peptide (VIP) and pituitary adenylyl cyclase activating peptide (PACAP) have pulmonary vasodilatory and positive inotropic effects via receptors VPAC1 and VPAC2, which possess a similar affinity for both peptides, and PAC1, a PACAP-preferring receptor. VIP is a promising option for PH treatment; however, various physiological effects of VIP have limited its clinical use. We investigated the effects of VPAC1 and VPAC2 selective agonists VIP and PACAP to explore more appropriate means of treatment for PH. We examined hemodynamic changes in right ventricular systolic pressure (RVSP), systemic blood pressure (SBP), total pulmonary resistance index (TPRI), total systemic resistance index, and cardiac index (CI) in response to their agonists with monocrotaline (MCT)-induced PH and explored involvement of VIP/PACAP expression and receptors in PH. Sprague-Dawley rats were divided into the MCT group (administered MCT 60 mg/kg) and control group. In MCT-induced PH, decreased VIP and PACAP were associated with upregulation of VPAC1, VPAC2, and PAC1 in lung tissues. Intravenous injection of VPAC2-selective agonist BAY 55-9837 and VIP, but not [Ala(11,22,28)]VIP, improved the CI. The decrease in SBP with VPAC2 agonist was significantly less than that in the control. Although they decreased SBP, these agonists hardly affected RVSP in the control. Activation of VPAC2 receptor with BAY 55-9837 effectively improved RVSP, TPRI, and CI in MCT-induced PH, suggesting a VPAC2 agonist as a possible promising treatment for PH.

Association of salivary cortisol levels and later depressive state in elderly people living in a rural community: a 3-year follow-up study.

BACKGROUND: Late-life depressive disorder is becoming an important issue in health economics in the world, as it has been reported to be one of major risk factors for incidence of dementia. Identification of predictive markers associated with depression in later life is therefore of high priority in public health. The aim of the study was to examine the association of salivary cortisol levels with a later depressive state in elderly healthy people living in a rural Japan community. METHODS: Salivary cortisol levels were measured in 68 elderly healthy people (24 men; 44 women) followed by completion of the BDI, MMSE, and FAB from 2004 to 2006. The same cohort underwent BDI again from 2007 to 2009. RESULTS: In healthy elderly women subject, a significant positive correlation was found between salivary cortisol levels at baseline and BDI scores at follow-up, but not at baseline. Salivary cortisol levels at baseline were not correlated with the score of either MMSE or FAB. When the cut-off point of BDI scores were set at 20/21, logistic regression analyses revealed that salivary cortisol levels at baseline had a significant positive relationship with a later depressive state. Age and gender were also significantly related with a later depressive state. LIMITATIONS: The present study involves small number of participants. CONCLUSIONS: Higher salivary cortisol levels were associated with a later depressive state in elderly healthy women living in rural community. Salivary cortisol might be a predictive marker for a later depressive state in elderly women.

Multicentre population-based dementia prevalence survey in Japan: a preliminary report.

Community-based surveys were performed in seven rural areas in Japan to investigate the prevalence of dementia and illnesses causing dementia. A total of 5431 elderly subjects were selected based on census data from 1 October 2009. In total, 3394 participants were examined (participation rate: 62.5%), and 768 dementia cases and 529 mild cognitive impairment cases were identified. Of the illnesses causing dementia, Alzheimer's disease was the most frequent (67.4%), followed by vascular dementia (18.9%), dementia with Lewy body disease (4.6%), mixed dementia (4.2%) and other illnesses. The prevalence of dementia according to 5-year age strata between 65 and 99 years was 5.8-77.7% among the participants. The prevalence of dementia in this study was higher than in previous reports in Japan and other countries. To verify the upward trend of dementia prevalence and its background factors, we have scheduled surveys for three other urban areas in 2011-2012.

Association of saliva 3-methoxy-4-hydroxyphenylglycol levels and a later depressive state in older subjects living in a rural community: 3-year follow-up study.

OBJECTIVE: The aim of the study was to examine the association of saliva levels of 3-methoxy-4-hydroxyphenylglycol (sMHPG) with a later depressive state in older people living in a rural community. METHODS: Baseline sMHPG levels were measured in 214 older subjects followed by completion of the Beck Depression Inventory (BDI) from 2004 to 2006 (time A). The same cohort underwent BDI again from 2007 to 2009 (time B). RESULTS: One hundred forty-four subjects (44 men, 100 women) were reassessed by the BDI. Baseline sMHPG levels in men with a BDI score of ≤9 at time A and a BDI score of ≥10 at time B were significantly higher than those in men with a BDI score of ≤9 at times A and B. In men, there was a significant correlation between baseline sMHPG levels and BDI score at time B (r = 0.40, p = 0.007) but not at time A (r = 0.29, p = 0.06). This association was not significant in women. CONCLUSION: These data indicate that high sMHPG levels at time A could be associated with a later depressive state in older men living in a community.

Baseline saliva level of 3-methoxy-4-hydroxyphenylglycole (MHPG) associates with a consequent cognitive decline in non-demented elderly subjects: three-years follow-up study.

The aim of the study was to explore the relation between saliva level of 3-methoxy-4-hydroxy-phenylglycol (MHPG) and a later cognitive decline in non-demented elderly subjects. We have reported that sMHPG in 214 elderly subjects living in the community (age 74.5±5.9years) was associated with scores on the Mini-Mental State Examination (MMSE) and the Frontal Assessment Battery (FAB) in 2004 to 2006 (Time A). The same cohort underwent these cognitive tests again from 2007 to 2009 (Time B). The cognitive function of the 147 of 214 subjects could be reassessed by the same cognitive tests. The score on the FAB, but not the MMSE, was significantly reduced at Time B (14.6±2.6) compared with that of Time A (15.2±1.9). There was a significant negative correlation between the baseline sMHPG and the changes in the FAB score subtracted from Time B to Time A or the scores on the FAB at Time B in men, but not at Time A. These correlations were not found in women. These data indicate that high sMHPG might be associated with subsequent cognitive decline assessed by the FAB in non-demented elderly men living in the community.

Actions of cathepsins on troponin T during postmortem aging of porcine muscle.

In this study, we examined the contribution of the cathepsins (cathepsin D and crude cathepsins containing cathepsins B and L) to troponin T degradation during postmortem aging. The action of cathepsin D on troponin T was examined at various pHs (pH 4.0-6.5). The degradation of intact troponin T was observed at pH 4.0, but not observed at pH 5.5 and 6.5. As a result of the degradation of troponin T, the 30 kDa fragment was not generated in any pH condition. The action of the crude cathepsins on troponin T was also examined at various pHs (pH 4.0-6.5). The intact troponin T was degraded at pH 4.0 and the 30 kDa fragments were observed. These 30 kDa fragments disappeared during further incubation. On the other hand, at pH 5.5 and 6.5, the intact troponin T was degraded and the 30 kDa fragment was accumulated. These results suggested that the cathepsin D scarcely contributed to the degradation of troponin T during postmortem aging, but crude cathepsins containing cathepsins B and L were partially involved in the degradation of troponin T and the generation of 30 kDa fragments.

Relationship between saliva level of 3-methoxy-4-hydroxyphenylglycol and mental health in the elderly general population.

AIMS: A large number of studies on the monoamine systems in Alzheimer's disease (AD) have found abnormalities of the noradrenergic system in the brain, but there has been no report concerning the relationship between noradrenergic activity and cognitive function in elderly living in a community. The aim of the present study was to explore the relationship between saliva level of 3-methoxy-4-hydroxyphenylglycol (sMHPG) and mental health in this population. METHODS: The study was to examine the relationship between sMHPG and performance on the Mini-Mental State Examination (MMSE), Frontal Assessment Battery (FAB), and Beck Depression Inventory (BDI) in 213 elderly people living in the local community. RESULTS: sMHPG in female subjects was positively correlated with age (r = 0.24, P = 0.003) and negatively correlated with scores on the MMSE (r = -0.26, P = 0.0016) and FAB (r = -0.19, P = 0.024), even after controlling for the effect of age (MMSE r = -0.20, P = 0.013). Notably, sMHPG was correlated with the pentagon drawing score (P = 0.0008) of MMSE. sMHPG was significantly correlated with BDI score in male subjects, but negatively correlated in female subjects. A gender difference was found in the relationship between the sMHPG and BDI score. CONCLUSION: The measurement of sMHPG may be a useful marker of mental health in elderly community-dwelling subjects.

Development of multilocus single strand conformation polymorphism (MLSSCP) analysis of virulence genes of Listeria monocytogenes and comparison with existing DNA typing methods.

Development of rapid and simple typing methods is required for analyzing the distribution and contamination routes of food-borne pathogens. We established a simple typing method for Listeria monocytogenes using MLSSCP (Multilocus Single Strand Conformation Polymorphism) analysis. Four virulence genes, hlyA, iap, actA and inlB were amplified by PCR, digested with endonucleases and applied to gels for SSCP. As banding patterns have been shown to reflect even a single nucleotide difference, this method has a potential discriminatory power comparable to that of sequencing analysis. The 64 strains isolated from five meat processing plants were divided into 18 groups by this MLSSCP. Additionally, clustering obtained with this method showed strong correspondence with phylogenetic lineages I and II, and was achieved with much less expenditure in time and cost than is required for other methods, such as MLST. The validity of the MLSSCP lineage classification was confirmed by PFGE, AFLP and ribotyping results. This newly developed MLSSCP method is suitable when obtaining accurate results quickly and simply is crucial.