Properties of hemolysin and protease produced by Aeromonas trota.

We examined the properties of exotoxins produced by Aeromonas trota (A. enteropelogenes), one of the diarrheagenic species of Aeromonadaceae. Nine of 19 A. trota isolates that grew on solid media containing erythrocytes showed hemolytic activity. However, the hemolytic activities of the culture supernatants of these hemolytic strains of A. trota were markedly lower than those of A. sobria when cultured in liquid medium, and the amount of hemolysin detected by immunoblotting using antiserum against the hemolysin produced by A. sobria was also low. A mouse intestine loop assay using living bacterial cells showed that A. trota 701 caused the significant accumulation of fluid, and antiserum against the hemolysin produced suppressed the enterotoxic action of A. trota 701. These results indicated that A. trota 701 was diarrheagenic and the hemolysin produced was the causative agent of the enterotoxic activity of A. trota. The hemolysin in A. sobria was previously shown to be secreted in a preform (inactive form) and be activated when the carboxy-terminal domain was cleaved off by proteases in the culture supernatant. Since mature hemolysin was detected in the culture supernatants of A. trota, we analyzed the extracellular protease produced by A. trota. Fifteen of 19 A. trota isolates that grew on solid media containing skim milk showed proteolytic activity. We subsequently found that most A. trota isolates possessed the serine protease gene, but not the metalloprotease gene. Therefore, we determined the nucleotide sequence of the serine protease gene and its chaperone A. trota gene. The results obtained revealed that the deduced amino acid sequences of serine protease and the chaperone were homologous to those of A. sobria with identities of 83.0% and 75.8%, respectively

Development of the Japanese Core Outcome Measures Index (COMI): cross-cultural adaptation and psychometric validation

Abstract Background The patient-rated Core Outcome Measures Index (COMI) assesses the multidimensional impact of back problems on the sufferer. The brevity and comprehensibility of the tool make it practical for use in clinical and research settings. Although the COMI has been cross-culturally adapted in various languages worldwide, there is currently no Japanese version. The aim of this study was to develop a Japanese version of the COMI by: (1) performing a cross-cultural adaptation of the English version and (2) evaluating the psychometric properties of the Japanese version of the COMI in Japanese volunteers with chronic back problems. Methods The English version of the COMI was cross-culturally adapted for the Japanese language using established guidelines. The pre-final version was pilot-tested in five Japanese-speaking patients with low back pain (LBP) and a history of spine surgery. The psychometric properties of the Japanese COMI were tested in a group of 1052 individuals with chronic LBP (LBP ≥3 months), aged 20–69 years, who were recruited through a web-based survey. The psychometric properties that were evaluated included convergent and known-group validity, using the following reference questionnaires: EuroQol 5 Dimension, Roland Morris Disability Questionnaire, Short Form 8™ Health Survey, and the Keele STarT Back Screening Tool. Results The pre-final version of the cross-culturally adapted Japanese COMI was completed without any major problems of understanding or acceptability. For the evaluation of its psychometric properties, tests for convergent validity showed moderate correlations between COMI items and the respective reference questionnaires for symptom-specific well-being [− 0.33–−0.48] and disability domains [0.48] and strong correlations (> 0.5) for the other domains and the COMI summary score. The analysis of known-group validity showed a linear trend for the COMI score in relation to prognostic risk (P < 0.001). Conclusions The Japanese COMI retained conceptual equivalence to the original using comprehensible and acceptable Japanese expressions. We developed a Japanese version of the COMI that displayed qualities that support its convergent and known-group validity. The availability of a Japanese version of the COMI should allow for improved documentation of the care provided to patients with back problems

Enterotoxic activity of <i>A. trota</i> 701.

<p>Enterotoxic activity was elucidated using the mouse intestinal loop test. A total of 3×10⁷ cells of <i>A. trota</i> 701 was mixed with either pre-immunization serum (white bar) or anti-ALH serum (black bar), and ingested into the mice intestinal loop. When the accumulation of fluid induced by the sample was more than 0.2 g/cm, the sample was considered to be enterotoxigenic. One group consisted of four mice.</p

Immunodetection of hemolysins produced by <i>A. sobria</i> and <i>A. trota</i>.

<p><i>A. sobria</i> 288, <i>A. trota</i> 701, and <i>A. trota</i> ATCC49657 were cultivated in NB medium at 37°C. A portion of the culture was collected at the period indicated and the culture supernatant and cell lysate were prepared as described in the text. Each SDS sample, which was equivalent to 50 µL culture, was applied to the lane (A). After SDS-PAGE, hemolysin was detected in each lane using anti-ALH antiserum, as described in the text. Larger amounts of SDS samples, equivalent to 250 µL culture, were applied to the lanes of SDS-PAGE for clearer detection (B). The sample containing pre-ALH and ALH was applied to lane P as a positive control. The upper band in lane P is pre-ALH and lower band is mature ALH.</p

Deduced amino acid sequence of serine protease and <i>A. trota</i> chaperone.

<p>The sequence (A) is the deduced amino acid sequence of serine protease and the sequence (B) is that of <i>A. trota</i> chaperone. Amino acid sequences are numbered as 1 from the initiator Met, and the numbers on the right side of the amino acid sequences indicate the number of amino acid residues from A of the initiation Met. The nucleotide sequence encoding these genes was deposited in GenBank (Accession No. KF914659). Underlined sections represent the amino acid sequence used to make anti-serine protease peptide antiserum.</p

Detection of the protease genes of <i>A. trota</i> by colony hybridization using specific probes.

<p>Each strain was inoculated on the NA plate and incubated at 37°C for 24 h. Bacterial colonies were transferred onto the Hybond-N⁺ membrane, and bacterial DNAs were baked. The colony reacted with specific probes for either the serine protease gene (A) or metalloprotease gene (B), as described in the text. The table on the right side of the figure shows the names of the strains used.</p

Immunodetection of serine protease in the culture supernatants of <i>A. sobria</i> and <i>A. trota</i>.

<p><i>A. sobria</i> 288, <i>A. trota</i> 701, and <i>A. trota</i> ATCC49657 were cultivated in NB medium at 37°C. A portion of the culture was collected at the period indicated and the culture supernatant was prepared as described in the text. Each SDS sample, which was equivalent to 50 µL culture, was applied to the lane. After SDS-PAGE, serine protease was detected in each lane using anti-serine protease peptide antiserum, as described in the text. The sample containing <i>A. sobria</i> serine protease was applied to lane P as a positive control. The band along with the arrow indicated the <i>A. sobria</i> serine protease with a molecular size of 64 kDa.</p

Cultivation of <i>A. trota</i> on agar medium containing erythrocytes or skim milk.

<p>Each strain was cultivated in nutrient broth for 20 µL of culture solutions were dropped on each agar medium. After inoculation, these plates were incubated at 37°C for 24 h. Hemolytic activity (A) and proteolytic activity (B) were assessed by the appearance of a transparent zone around the bacteria on each plate, respectively. The table at the right side of the figure shows the names of the strains used.</p

Clinical Effect of Lenvatinib Re-Administration after Transcatheter Arterial Chemoembolization in Patients with Intermediate Stage Hepatocellular Carcinoma

The present study clarified the prognosis of intermediate-stage hepatocellular carcinoma (HCC) patients who received lenvatinib (LEN) followed by transcatheter arterial chemoembolization (TACE) on demand. We retrospectively evaluated 88 intermediate-stage HCC patients who received LEN. The median age was 74 (range: 47–92) years old, 67 patients were male, and 82 were classified as Child-Pugh A. LEN was administered until disease progression or discontinuation due to adverse events (AEs). The mean duration of LEN treatment was 7.0 months. The response and disease control rates were 51.1% and 89.8%, respectively. The median progression-free survival and overall survival (OS) after the initiation of LEN were 6.8 months and 29.9 months, respectively. The OS in patients for whom LEN was re-administered after TACE (TACE-LEN) was better than that in patients who received other therapies (e.g., only TACE, TACE-other therapy, or only other therapy) even with propensity score matching (p = 0.008). A Cox proportional hazard analysis showed that TACE-LEN was most strongly associated with the OS (hazard ratio: 0.083, 95% confidence interval: 0.019–0.362, p = 0.001). LEN was administered for approximately 11.1 months after TACE. In intermediate-stage HCC patients who can tolerate LEN without discontinuation due to AEs, TACE-LEN may prolong the prognosis