Modelling the invasion of human small intestinal epithelial-like cells by Salmonella enterica Typhimurium and Listeria monocytogenes using Bayesian inference.

To develop a mechanistic bacterial dose-response model, based on the concept of Key Events Dose-Response Framework (KEDRF), this study aimed to investigate the invasion of intestinal model cells (Caco-2) by Salmonella Typhimurium and Listeria monocytogenes and described the behaviour of both pathogens as a mathematical model using Bayesian inference. Monolayer-cultured Caco-2 cells (approximately 105 cells) were co-cultured with various concentrations (103 -107 colony forming unit [CFU] ml-1 ) of Salm. Typhimurium and L. monocytogenes for up to 9 h to investigate the invasion of the pathogens into the Caco-2 cells. While an exposure of ≥103 CFU ml-1 of Salm. Typhimurium initiated the invasion of Caco-2 cells within 3 h, much less exposure (102 CFU ml-1 ) of L. monocytogenes was sufficient for invasion within the same period. Furthermore, while the maximum number of invading Salm. Typhimurium cells reached by approximately 103 CFU cm-2 for 6-h exposure, the invading maximum numbers of L. monocytogenes cells increased by approximately 106 CFU cm-2 for the same exposure period. The invasion kinetics of both the pathogens was successfully described as an asymptotic exponential mathematical model using Bayesian inference. The developed pathogen invasion model allowed the estimation of probability of Salm. Typhimurium and L. monocytogenes infection, based on the physiological natures of digestion process, which was comparable to the published dose-response relationship. The invasion models developed in the present study will play a key role in the development of an alternative pathogen dose-response model based on KEDRF concept.

Seasonality of coastal upwelling trends in the Mauritania-Senegalese region under RCP8.5 climate change scenario.

The Mauritania-Senegalese upwelling region (MSUR), the southernmost region of the Canary current upwelling system, is well-known for its coastal productivity and the key role it plays in enriching the oligotrophic open ocean through the offshore transport of the upwelled coastal waters. The great ecological and socio-economic importance makes it necessary to evaluate the impact of climate change on this region. Hence, our main objective is to examine the climate change signal over the MSUR with a high resolution regional climate system model (RCSM) forced by the Earth system model MPI-ESM-LR under RCP8.5 scenario. This RCSM has a regional atmosphere model (REMO) coupled to a global ocean model (MPIOM) with high-resolution in the MSUR, which allows us to evaluate the wind pattern, the ocean stratification, as well as the upwelling source water depth, while maintaining an ocean global domain. Under RCP8.5 scenario, our results show that the upwelling favourable winds of the northern MSUR are year-round intensified, while the southern MSUR presents a strengthening in winter and a weakening in March-April. Along with changes in the wind pattern, we found increased ocean stratification in the spring months. In those months southern MSUR presents a shallowing of the upwelling source water depth associated to changes in both mechanisms. However, in winter the whole MSUR shows a deepening of the upwelling source water depth due to the intensification of the upwelling favourable winds, with the increased ocean stratification playing a secondary role. Our results demonstrate the need to evaluate the future evolution of coastal upwelling systems taking into account their latitudinal and seasonal variability and the joint contribution of both mechanisms.

Evaluation of thermal inactivation of Escherichia coli using microelectrode ion flux measurements with osmotic stress.

AIMS: To elucidate the potential use of microelectrode ion flux measurements to evaluate bacterial responses to heat treatment. METHODS AND RESULTS: Escherichia coli K12 was used as a test bacterium to determine whether various heat treatments (55-70°C for 15 min) affected net ion flux across E. coli cell membranes using the MIFE™ system to measure net K(+) fluxes. No difference in K(+) fluxes was observed before and after heat treatments regardless of the magnitude of the treatment. Applying hyperosmotic stress (3% NaCl w/v) during flux measurement led to a net K(+) loss from the heat-treated E.coli cells below 65°C as well as from nonheated cells. In contrast, with E. coli cells treated at and above 65°C, hyperosmotic stress disrupted the pattern of K(+) flux observed at lower temperatures and resulted in large flux noise with random scatter. This phenomenon was particularly apparent above 70°C. Although E. coli cells lost the potential to recover and grow at and above 62°C, K(+) flux disruption was not clearly observed until 68°C was reached. CONCLUSIONS: No changes in net K(+) flux from heat-stressed E. coli cells were observed directly as a result of thermal treatments. However, regardless of the magnitude of heat treatment above 55°C, loss of viability indicated by enrichment culture correlated with disrupted K(+) fluxes when previously heated cells were further challenged by imposing hyperosmotic stress during flux measurement. This two-stage process enabled evaluation of the lethality of heat-treated bacterial cells within 2 h and may be an alternative and more rapid method to confirm the lethality of heat treatment. SIGNIFICANCE AND IMPACT OF THE STUDY: The ability to confirm the lethality of thermal treatments and to specify minimal time/temperature combinations by a nonculture-dependent test offers an alternative system to culture-based methods.

Formation of a catalytically active dimer by tRNA(Val)-driven short ribozymes.

A minizyme is a hammerhead ribozyme with a short oligonucleotide linker instead of stem/loop II. Minizymes with low activity as monomers form active dimeric structures with a common stem. We explored the use of dimeric minizymes as gene-inactivating agents by placing minizymes under the control of a tRNA(Val) promoter. The tRNA(Val) portion of the transcript did not hinder dimerization as the tRNA-embedded minizyme formed an active dimeric structure. The cleavage activity of this minizyme that had been expressed either in vitro or in HeLa cells was almost one order of magnitude higher than that of the tRNA(Val)-embedded conventional hammerhead ribozyme. The tRNA(Val)-driven minizyme inhibited reporter gene activity (95%) whereas the tRNA(Val)-driven hammerhead ribozyme resulted in approximately 55% inhibition.

Significantly higher activity of a cytoplasmic hammerhead ribozyme than a corresponding nuclear counterpart: engineered tRNAs with an extended 3' end can be exported efficiently and specifically to the cytoplasm in mammalian cells.

Hammerhead ribozymes were expressed under the control of similar tRNA promoters, localizing transcripts either in the cytoplasm or the nucleus. The tRNA(Val)-driven ribozyme (tRNA-Rz; tRNA with extra sequences at the 3' end) that has been used in our ribozyme studies was exported efficiently into the cytoplasm and ribozyme activity was detected only in the cytoplasmic fraction. Both ends of the transported tRNA-Rz were characterized comprehensively and the results confirmed that tRNA-Rz had unprocessed 5' and 3' ends. Furthermore, it was also demonstrated that the activity of the exported ribozyme was significantly higher than that of the ribozyme which remained in the nucleus. We suggest that it is possible to engineer tRNA-Rz, which can be exported to the cytoplasm based on an understanding of secondary structures, and then tRNA-driven ribozymes may be co-localized with their target mRNAs in the cytoplasm of mammalian cells.

Intercellular cell adhesion molecule-1 regulates lymphocyte movement into intestinal microlymphatics of rat Peyer's patches.

The objective of this study was to determine whether specific adhesion molecules modulate lymphocyte movement from Peyer's patches into intestinal microlymphatics. The fluorochrome acridine orange was injected via a micropipette into Peyer's patches to fill lymphatics. The flux of labeled lymphocytes into intestinal microlymphatics was monitored with intravital fluorescence microscopy. The lymphatic microvessels in the perifollicular area of Peyer's patches were filled with lymphocytes, most of which remained within the lymphatics. Some lymphocytes became detached and were drained into intestinal lymph. Administration of antibodies directed against ICAM-1 significantly increased lymphocyte flux into interfollicular lymphatics. The immunohistochemical study showed intense ICAM-1 expression on the lymphocytes densely packed in the lymphatics surrounding follicles in Peyer's patches. A large number of lymphocytes are normally sequestered in the lymphatic network of Peyer's patches. This sequestration of lymphocytes is largely mediated by ICAM-1-dependent cell-cell interactions.

A simple assay system for examination of the inhibitory potential in vivo of decoy RNAs, ribozymes and other drugs by measuring the Tat-mediated transcription of a fusion gene composed of the long terminal repeat of HIV-1 and a gene for luciferase.

Nucleic acid-based drugs, including antisense RNA and DNA, ribozymes and decoys appear to have potential for the suppression of the expression of specific genes. To allow the examination of the potential of such agents in vivo as anti-HIV drugs in standard laboratories, where facilities for handling live virions are not available, we constructed a simple assay system (HIV-1 model) that allows measurement of the extent of inhibition of Tat-mediated transcription of HIV-1 by nucleic acid-based drugs and other agents. In cells that harbor a stable chimeric long terminal repeat (LTR)-Luc construct (a fusion gene consisting of the LTR of HIV-1 and the gene for luciferase), total luciferase activity in an aliquot of cell lysate is dose- and promoter-dependent on transfection with a Tat expression plasmid, reflecting the character of the LTR promoter of HIV. When HeLa cells were co-transfected with the Tat expression plasmid and another plasmid that encoded the U6 promoter or the promoter of the gene for tRNA(Val) linked to the trans-activating response (TAR) sequence, total luciferase activity was inhibited by 60 or 40%, respectively. The inhibition was also dependent on the dose of the TAR expression plasmid. These results demonstrate the usefulness of this simple assay system for detection of the efficacy of a decoy RNA or a ribozyme in vivo, without a requirement for HIV-infected cells, by measurement of luciferase activity in vitro.

Secondary diabetic retinopathy in chronic pancreatitis.

We found abnormal glucose tolerance curves in 45 of 47 patients with chronic pancreatitis and observed secondary diabetic retinopathy in eight of 45 cases showing slight changes in the fundus. Abnormal glucose tolerance curves were somewhat related to the exocrine dysfunction of the pancreas. Slightly abnormal glucose tolerance curves were observed frequently in patients with chronic pancreatitis, and both insulin and glucagon responses were decreased. We could not explain the cause of the low frequency of secondary diabetic retinopathy in pancreatic diabetes from the results of insulin and glucagon response tests.

Characterization of anti-irradiation-denatured ovalbumin monoclonal antibodies. Immunochemical and structural analysis of irradiation-denatured ovalbumin.

Five monoclonal antibodies (OVA-01, -02, -03, -04, -06) produced against irradiated ovalbumin were investigated in relation to the conformational change in the ovalbumin molecule induced by irradiation with Cobalt-60 gamma-rays. Four antibodies (OVA-01, -02, -04, -06) recognized both native and irradiated ovalbumin, but OVA-03 reacted only with irradiated ovalbumin. These antibodies were classified by modified competitive ELISA, and their K(d) values were determined by the Klotz equation. Epitope analyses were also performed on OVA-03 using CNBr-cleaved peptide fragments from ovalbumin, and it was confirmed that OVA-03 bound to the fragment corresponding to residues Val173-Met196 of the ovalbumin molecule that consists of internal beta-sheet strand 3A and helix F1 containing one open turn. These results demonstrate that dramatic conformational changes in proteins can be induced or that some tertiary or secondary structures can be broken down by gamma-ray irradiation, producing new antigenic sites.

Prediction of microbial growth in fresh-cut vegetables treated with acidic electrolyzed water during storage under various temperature conditions.

Effects of storage temperature (1, 5, and 10 degrees C) on growth of microbial populations (total aerobic bacteria, coliform bacteria, Bacillus cereus, and psychrotrophic bacteria) on acidic electrolyzed water (AcEW)-treated fresh-cut lettuce and cabbage were determined. A modified Gompertz function was used to describe the kinetics of microbial growth. Growth data were analyzed using regression analysis to generate "best-fit" modified Gompertz equations, which were subsequently used to calculate lag time, exponential growth rate, and generation time. The data indicated that the growth kinetics of each bacterium were dependent on storage temperature, except at 1 degrees C storage. At 1 degrees C storage, no increases were observed in bacterial populations. Treatment of vegetables with AcEW produced a decrease in initial microbial populations. However, subsequent growth rates were higher than on nontreated vegetables. The recovery time required by the reduced microbial population to reach the initial (treated with tap water [TW]) population was also determined in this study, with the recovery time of the microbial population at 10 degrees C being <3 days. The benefits of reducing the initial microbial populations on fresh-cut vegetables were greatly affected by storage temperature. Results from this study could be used to predict microbial quality of fresh-cut lettuce and cabbage throughout their distribution.

Decontamination of lettuce using acidic electrolyzed water.

The disinfectant effect of acidic electrolyzed water (AcEW), ozonated water, and sodium hypochlorite (NaOCl) solution on lettuce was examined. AcEW (pH 2.6; oxidation reduction potential, 1140 mV; 30 ppm of available chlorine) and NaOCl solution (150 ppm of available chlorine) reduced viable aerobes in lettuce by 2 log CFU/g within 10 min. For lettuce washed in alkaline electrolyzed water (AIEW) for 1 min and then disinfected in AcEW for 1 min, viable aerobes were reduced by 2 log CFU/g. On the other hand, ozonated water containing 5 ppm of ozone reduced viable aerobes in lettuce 1.5 log CFU/g within 10 min. It was discovered that AcEW showed a higher disinfectant effect than did ozonated water significantly at P < 0.05. It was confirmed by swabbing test that AcEW, ozonated water, and NaOCI solution removed aerobic bacteria, coliform bacteria, molds, and yeasts on the surface of lettuce. Therefore, residual microorganisms after the decontamination of lettuce were either in the inside of the cellular tissue, such as the stomata, or making biofilm on the surface of lettuce. Biofilms were observed by a scanning electron microscope on the surface of the lettuce treated with AcEW. Moreover, it was shown that the spores of bacteria on the surface were not removed by any treatment in this study. However, it was also observed that the surface structure of lettuce was not damaged by any treatment in this study. Thus, the use of AcEW for decontamination of fresh lettuce was suggested to be an effective means of controlling microorganisms.

Impaired left ventricular rapid filling during exercise in patients with hypertrophic cardiomyopathy.

The influence of exercise on left ventricular diastolic filling was evaluated in 14 patients with hypertrophic cardiomyopathy (HCM) and 14 normal controls (NC) by dynamic exercise echocardiography. Using X-Y digitizer and computer, normalized peak rate of change of the left ventricular dimension during systole (pVs) and the rapid filling phase (pVd) were determined from the left ventricular echocardiograms at rest and during exercise when heart rate reached 100 beats/min. At rest and during exercise, pVs was significantly higher in HCM (3.2 +/- 0.4/s at rest, 4.3 +/- 1.4/s during exercise) than in NC (2.4 +/- 0.5/s at rest, 3.0 +/- 0.4s during exercise) (p less than 0.001, p less than 0.001, respectively), but pVd in HCM (4.2 +/- 1.0/s at rest, 5.8 +/- 1.0/s during exercise) was not significantly different from that in NC (4.1 +/- 1.0/s at rest, 6.0 +/- 0.7/s during exercise). The ratio of pVd to pVs (pVd/pVs) in HCM did not show significant increment during exercise (1.35 +/- 0.38 to 1.43 +/- 0.35), though that ratio in NC was significantly increased by exercise (1.67 +/- 0.22/s to 1.97 +/- 0.19/s, p less than 0.001). There was no correlation between pVd and the degree of left ventricular hypertrophy. These results suggest that diastolic reserve to exercise is depressed in HCM and that other factors besides left ventricular hypertrophy may account for diastolic abnormality.

Absent myocardial accumulation of two different radioiodinated pentadecanoic acids.

This article presents two cases with preserved myocardial 201Tl uptake and absent uptake of two kinds of radioiodinated fatty acids: iodine-123-labeled 15-(p-iodophenyl)-3-(R,S)-methylpentadecanoic acid (BMIPP) and iodine-123-labeled 15-(p-iodophenyl)-9-(R,S)-methylpentadecanoic acid (9MPA). Although coronary angiography showed no stenotic lesion and left ventriculography revealed no wall motion abnormality, no myocardial uptake of BMIPP and 9MPA was observed in the first case. In the second case, no myocardial accumulation was recognized even in the initial phase of dynamic SPECT acquired soon after the injection of 9MPA. The results suggest that the non-visualized myocardium was not specific for BMIPP imaging and that rather than the early back diffusion of the tracers from the myocardium, abnormality of the myocardial cell membrane was a possible mechanism accounting for the phenomenon.

Effect of physical activity on the distensibility of the aortic wall in healthy males.

To study the effect of physical activity level on the distensibility of the human aortic wall, aortic pulse wave velocity (APWV) was estimated in 139 healthy male subjects (19-67 years) and was related to the energy expenditure by habitual physical exercise (physical activity index: PAI), which was evaluated by a 7-day total activity recall. The subjects consisted of 56 fun runners (runner group) and 83 general subjects, who were divided into 25 active subjects (active group: PAI > or = 1,500 kcal/week) and 58 sedentary subjects (sedentary group: PAI < 1,500 kcal/week). The APWV index (APWVI: standardized APWV by the diastolic blood pressure) was found to be positively correlated with age and was negatively correlated with PAI. The age-adjusted APWVI of the runner group was significantly lower than that of the active and sedentary groups. The age-adjusted APWVI was also significantly lower in the active group than in the sedentary group. These results suggest that increased physical activity may retard the age-dependent loss of arterial distensibility in humans, in proportion to the amount and/or intensity of exercise.

Squamous cell carcinoma arising in chronic perianal pyoderma a case report and review of Japanese literature.

We report a rare case of squamous cell carcinoma developing from fistules of chronic perianal pyoderma in a 49-year-old Japanese man. He first noticed an abscess and nodule on his buttocks and perianal area 21 year previously (at the age of 28); the fistules formed later. These fistules were surgically removed, and an artificial anus was constructed 14 years ago (at the age of 35) in our hospital, when a histopathological examination revealed no malignant changes. However, he was recently admitted to our hospital with arterial bleeding from the ulcer of the buttock. On admission, the histological diagnosis of the ulcer was well differentiated squamous cell carcinoma. Wide local excision of the ulcer and scar tissue, including the sacrum, was performed. The defect was covered with a left latissimus dorsi flap and skin graft. He received radiation therapy after the operation. However, he died of cachexia and pneumonia. This case indicated that the CPP would better have been treated with wide excision before the development of SCC. Therefore, we recommend careful follow-up of patients affected by CPP and repeated biopsies of the lesion, particularly when the condition is severe, longstanding, and extensive. We discussed the term "CPP" and reviewed 22 cases of SCC arising in CPP reported in the Japanese literature.

A case of triple extramammary Paget's disease.

We report a 74-year-old Japanese man with triple extramammary Paget's disease; the genital and both of the axillar regions were simultaneously involved. Literature review revealed that 27 cases of triple extramammary Paget's disease have been reported in Japan, but there are no reports of triple extramammary Paget's disease from countries other than Japan, although 2 cases of double extramammary Paget's disease are reported. In all 28 cases, including our case, the genital lesion preceded the axillary lesions. All cases except one were male and only the exceptional female case was reported to have an the invasive tumor in the dermis. We speculate that extramammary Paget's disease may appear multi-centrically.

Assessment of cellular proliferation of sebaceous neoplasms by AgNOR counts and immunohistochemical demonstrations of PCNA and Ki-67.

We assessed cellular proliferation of sebaceous neoplasms by AgNOR counts and the immunohistochemical demonstration of proliferating cell nuclear antigen (PCNA) and Ki-67, using formalin-fixed and paraffin-embedded tissue specimens. We used three categories of sebaceous neoplasms: four cases of sebaceoma, three cases of basal cell carcinoma with sebaceous differentiation (BCSD), and seven cases of sebaceous carcinoma (SC). Significant differences were noted between SC and non-SC tumors (sebaceoma and BCSD) in AgNOR counts and semi-quantitative grading of PCNA and Ki-67 labelling indices (P < 0.01). When a cut-off value of 6 was chosen, the AgNOR value discriminated SC from non-SC tumors with high specificity and sensitivity. When a cut-off value of 25% was chosen, PCNA and Ki-67 labelling indices also discriminated between these tumors. Significant differences were not observed between sebaceoma and BCSD with PCNA and Ki-67 labelling indices. AgNOR counts of BCSD were a little higher than those of sebaceoma, but the number of cases was too small to perform statistical assessment. We consider AgNOR counts and semi-quantitative grading of PCNA and Ki-67 labelling indices to be useful in differentiating SC from BCSD and sebaceoma.

E-cadherin expression in skin tumors using an AMeX immunohistostaining method.

The AMeX (acetone-methylbenzoate-xylene) method results in good preservation of tissue and morphological details, almost equivalent to that of routinely processed formalin-fixed and paraffin-embedded tissue specimens, and of antigenicity equivalent to that of fresh frozen tissue specimens. It has been reported that the expression of the cell-cell adhesion molecule E-cadherin is often decreased in some types of carcinomas. A decrease in E-cadherin expression is associated with the invasive or metastatic potential of tumor cells. We immunohistochemically examined the expression of E-cadherin with anti-E-cadherin monoclonal antibody in various skin tumors (25 basal cell carcinomas, 11 squamous cell carcinomas, 9 keratoacanthomas, and 11 Bowen's disease) using the AMeX method and found that this method preserved antigenicity well without pretreatment. E-cadherin expression was decreased in 18.2% of squamous cell carcinomas and 33.3% of keratoacanthomas. On the other hand, it was preserved in almost all Bowen's disease and basal cell carcinomas. From the results of our study, we suggest that Bowen's disease and basal cell carcinoma do not have much metastatic potential due to retention of high levels of E-cadherin expression. We hope to apply the AMeX method to other immunohistochemical examinations because this is a very useful staining method.

An immunohistochemical study of E-cadherin expression in human squamous cell carcinoma of the skin: relationship between decreased expression of E-cadherin in the primary lesion and regional lymph node metastasis.

E-cadherin is a Ca(2+)-dependent, intercellular adhesion molecule that is specifically expressed in epithelial tissues and is essential for maintaining intercellular connections. It has been reported that E-cadherin expression of tumor cells is often decreased in some types of metastasizing carcinomas as compared with those without metastasis. We immunohistochemically examined the expression of E-cadherin with anti-E-cadherin monoclonal antibody and compared primary lesions of human squamous cell carcinoma of the skin (SCC) with regional lymph node metastasis to those without regional lymph node metastasis. Tumor samples from fifty-five cases of SCC (32 cases of SCC without metastasis and 23 cases with metastasis) were formalin-fixed, paraffin-embedded, and examined. E-cadherin was reduced or absent in 39 (70.9%) out of 55 cases of SCC, and in 21 (91.3%) of 23 cases with regional lymph node metastasis. Our results suggest that the decreased expression of E-cadherin in the primary lesion is correlated with regional lymph node metastasis in SCC and that it is more frequently correlated with well-differentiated than with poorly differentiated SCC. E-cadherin may be useful as a marker for metastatic potential in well-differentiated SCC.

Evaluation of left ventricular reserve in left ventricular diseases: non-invasive analysis of its determinants by dynamic exercise echocardiography.

To analyze the determinants of left ventricular (LV) reserve non-invasively, LV echocardiograms were taken during ergometer exercise in 52 LV disease patients (19 with hypertension [HT], 22 with aortic regurgitation [AR] and 11 with dilated cardiomyopathy [DCM] with various severities. In mild cases [HT], at exercise, fractional shortening (FS) increased, and at higher-grade exercise, an increase of LV enddiastolic diameter (Dd) was added to more increase of FS, to increase stroke volume (SV). In moderately severe cases [HT, AR or DCM], FS did not change or decreased at exercise. In severe cases [AR or DCM], FS was low at rest, and at exercise decreased further with an increase of LV systolic stress and no significant increase of Dd, resulting in a decrease of SV. The responses of LV parameters in volume-overload (AR) or dilated myocardial (DCM) hearts were poorer than in pressure-overload (HT) hearts, in moderately severe cases. In conclusion, exercise echocardiography can show non-invasively that the interrelationship among the determinants of LV reserve is different by the severities and the types of LV diseases, and this method is useful, especially in discriminating mild cases objectively.