Displaced Intra-Articular Calcaneal Fractures: Evaluation of Clinical and Radiological Outcome Following Open Reduction and Internal Fixation with Locking Branched Calcaneal Plate.

Background An introduction of the World Health Organization Surgical Safety Checklist (WHO SSC) is essential to promote surgical safety. Objective To obtain country-specific information regarding the checklist in a leading medical institution in Nepal. Method The present research was a cross-sectional study with a survey conducted among healthcare professionals working in the operation theatre at the Tribhuvan University Teaching Hospital (TUTH) in Kathmandu, Nepal. A questionnaire was distributed to 150 healthcare professionals working in the operating theatre. Responses to the questionnaire were analysed descriptively and regression analyses used to identify factors associated with awareness of the checklist. Result In total, 127 healthcare professionals participated in the study, of whom 118 (92.9%) had been aware of the WHO SSC. A substantial proportion of participants (108, 91.5%) were not satisfied with the prevailing practice whereby the checklist was not routinely used during surgery. Lack of appropriate training was the most prevalent barrier to the checklist use (72, 67.9%), followed by unwillingness of staff to use the checklist (54, 50.9%), and lack of experience (42, 39.7%). The mean score on the survey was 6.0 out of 10. Regarding the results of the regression model on survey scores, surgeons had higher scores compared to nurses (unadjusted coefficient 0.80, 95% CI 0.20-1.40). Conclusion Most of the healthcare professionals were aware of the WHO SSC, however multiple barriers to the checklist use were identified. It is important to establish an effective use of WHO SSC in the operation theatre.

Antimicrobial Stewardship Opportunities in Patients with Bacteremia Not Identified by BioFire FilmArray.

A subset of bacteremia cases are caused by organisms not detected by a rapid-diagnostics platform, BioFire blood culture identification (BCID), with unknown clinical characteristics and outcomes. Patients with ≥1 positive blood culture over a 15-month period were grouped by negative (NB-PC) versus positive (PB-PC) BioFire BCID results and compared with respect to demographics, infection characteristics, antibiotic therapy, and outcomes (length of hospital stay [LOS] and in-hospital mortality). Six percent of 1,044 positive blood cultures were NB-PC. The overall mean age was 65 ± 22 years, 54% of the patients were male, and most were admitted from home; fewer NB-PC had diabetes (19% versus 31%, P = 0.0469), although the intensive care unit admission data were similar. Anaerobes were identified in 57% of the bacteremia cases from the NB-PC group by conventional methods: Bacteroides spp. (30%), Clostridium (11%), and Fusobacterium spp. (8%). Final identification of the NB-PC pathogen was delayed by 2 days (P < 0.01) versus the PB-PC group. The sources of bacteremia were more frequently unknown for the NB-PC group (32% versus 11%, P < 0.01) and of pelvic origin (5% versus 0.1%, P < 0.01) compared to urine (31% versus 9%, P < 0.01) for the PB-PC patients. Fewer NB-PC patients received effective treatment before (68% versus 84%, P = 0.017) and after BCID results (82% versus 96%, P = 0.0048). The median LOS was similar (7 days), but more NB-PC patients died from infection (26% versus 8%, P < 0.01). Our findings affirm the need for the inclusion of anaerobes in BioFire BCID or other rapid diagnostic platforms to facilitate the prompt initiation of effective therapy for bacteremia.

Underestimation of COVID-19 cases in Japan: an analysis of RT-PCR testing for COVID-19 among 47 prefectures in Japan.

BACKGROUND: Under the unique Japanese policy to restrict reverse transcriptase-polymerase chain reaction (RT-PCR) testing against severe acute respiratory syndrome coronavirus 2, a nationwide number of its confirmed cases and mortality remains to be low. Yet the information is lacking on geographical differences of these measures and their associated factors. AIM: Evaluation of prefecture-based geographical differences and associated predictors for the incidence and number of RT-PCR tests for coronavirus disease 2019 (COVID-19). DESIGN: Cross-sectional study using regression and correlation analysis. METHODS: We retrieved domestic laboratory-confirmed cases, deaths and the number of RT-PCR testing for COVID-19 from 15 January to 6 April 2020 in 47 prefectures in Japan, using publicly available data by the Ministry of Health, Labour and Welfare. We did descriptive analyses of these three measures and identified significant predictors for the incidence and RT-PCR testing through multiple regression analyses and correlates with the number of deaths through correlation analysis. RESULTS: The median prefectural-level incidence and number of RT-PCR testing per 100 000 population were 1.14 and 38.6, respectively. Multiple regression analyses revealed that significant predictors for the incidence were prefectural-level population (P < 0.001) and the number of RT-PCR testing (P = 0.03); and those for RT-PCR testing were the incidence (P = 0.025), available beds (P = 0.045) and cluster infections (P = 0.034). CONCLUSION: Considering bidirectional association between the incidence and RT-PCR testing, there may have been an underdiagnosed population for the infection. The restraint policy for RT-PCR testing should be revisited to meet the increasing demand under the COVID-19 epidemic.

Genotyping of Edwardsiella ictaluri isolates in Japan using amplified-fragment length polymorphism analysis.

AIM: The major objective of the present study was to clarify genetic relationship of isolates of Edwardsiella ictaluri in Japan, which was first found from ayu Plecoglossus altivelis in Japanese rivers in 2007. METHODS AND RESULTS: Ten isolates of Edw. ictaluri in 2007-2008 from ayu and the 1 isolate from bagrid catfish Pelteobagrus nudiceps in Japan were subjected to amplified-fragment length polymorphism (AFLP) analysis. The strains isolated from catfish in United States (ATCC strains) or Indonesia were used as reference strains. The AFLP profiles were all the same among the isolates from Japan, while the polymorphic DNA bands were observed among the strains from United States or Indonesia. The isolates from Japan and Indonesia constituted a genogroup different from the ATCC strains on a dendrogram constructed from the AFLP profiles. CONCLUSION: No DNA polymorphisms were found among Japanese Edw. ictaluri isolates. SIGNIFICANCE AND IMPACT OF THE STUDY: A single clonality of the Edw. ictaluri isolates in Japan suggests the single source of the organism, and the infection in ayu is in the early stage of epidemics.

Large-scale production of UDP-galactose and globotriose by coupling metabolically engineered bacteria.

A large-scale production system of uridine 5'-diphospho-galactose (UDP-Gal) has been established by the combination of recombinant Escherichia coli and Corynebacterium ammoniagenes. Recombinant E. coli that overexpress the UDP-Gal biosynthetic genes galT, galK, and galU were generated. C. ammoniagenes contribute the production of uridine triphosphate (UTP), a substrate for UDP-Gal biosynthesis, from orotic acid, an inexpensive precursor of UTP. UDP-Gal accumulated to 72 mM (44 g/L) after a 21 h reaction starting with orotic acid and galactose. When E. coli cells that expressed the alpha1,4-galactosyltransferase gene of Neisseria gonorrhoeae were coupled with this UDP-Gal production system, 372 mM (188 g/L) globotriose (Galalpha1-4Galbeta1-4Glc), a trisaccharide portion of verotoxin receptor, was produced after a 36 h reaction starting with orotic acid, galactose, and lactose. No oligosaccharide by-products were observed in the reaction mixture. The production of globotriose was several times higher than that of UDP-Gal. The strategy of producing sugar nucleotides by combining metabolically engineered recombinant E. coli with a nucleoside 5'-triphosphate producing microorganism, and the concept of producing oligosaccharides by coupling sugar nucleotide production systems with glycosyltransferases, can be applied to the manufacture of other sugar nucleotides and oligosaccharides.

Oral CD103-CD11b+ classical dendritic cells present sublingual antigen and induce Foxp3+ regulatory T cells in draining lymph nodes.

Sublingual immunotherapy (SLIT) is a safe and efficient treatment for type 1 allergies; however, the underlying immunological mechanisms, particularly the phenotype of oral antigen-presenting cells (APCs) responsible for the induction of regulatory T (Treg) cells, remain unclear. We show here that the sublingual application of ovalbumin (OVA) induced antigen-specific Foxp3+ Treg cells in draining submandibular lymph nodes (ManLNs). Oral APCs were classified into macrophages, classical dendritic cells (cDCs), and Langerhans cells by flow cytometry. A major subset of oral cDCs with the CD103-CD11b+ phenotype showed retinoic acid (RA)-producing activity and converted naive CD4+ T cells to Foxp3+ Treg cells in a transforming growth factor-ß- and RA-dependent manner in vitro. In the ManLNs, migratory CD103-CD11b+ cDCs also showed RA-producing activity. After the sublingual application of fluorescent OVA, fluorescence was detected in oral macrophages in tissues, followed by migratory CD103-CD11b+ cDCs in ManLNs and migratory CD103-CD11b+ cDCs were the main APCs responsible for the induction of sublingual antigen-specific Treg cells. The transfer of OVA-SLIT-induced Treg cells suppressed the OVA-induced hypersensitivity response. These results suggest that oral CD103-CD11b+ cDCs transport sublingual antigens to draining ManLNs and induce antigen-specific Foxp3+ Treg cells, and, thus, provide a rationale for developing cDC-based therapeutic approaches in SLIT.

Purification of cohesive-end-producing restriction endonuclease from Bacillus subtilis G.

A new restriction endonuclease was partially purified from Bacillus subtilis G (IAM1247). This restriction endonuclease (endonuclease RBsuG) seems to produce cohesive ends at its cleavage site.

A microsatellite linkage map of rainbow trout (Oncorhynchus mykiss) characterized by large sex-specific differences in recombination rates.

We constructed a genetic linkage map for a tetraploid derivative species, the rainbow trout (Oncorhynchus mykiss), using 191 microsatellite, 3 RAPD, 7 ESMP, and 7 allozyme markers in three backcross families. The linkage map consists of 29 linkage groups with potential arm displacements in the female map due to male-specific pseudolinkage arrangements. Synteny of duplicated microsatellite markers was used to identify and confirm some previously reported pseudolinkage arrangements based upon allozyme markers. Fifteen centromeric regions (20 chromosome arms) were identified with a half-tetrad analysis using gynogenetic diploids. Female map length is approximately 10 M, but this is a large underestimate as many genotyped segments remain unassigned at a LOD threshold of 3.0. Extreme differences in female:male map distances were observed (ratio F:M, 3.25:1). Females had much lower recombination rates (0.14:1) in telomeric regions than males, while recombination rates were much higher in females within regions proximal to the centromere (F:M, 10:1). Quadrivalent formations that appear almost exclusively in males are postulated to account for the observed differences.

Whole microbial cell processes for manufacturing amino acids, vitamins or ribonucleotides.

The development of recombinant DNA technology has greatly expanded whole microbial cell processes for manufacturing amino acids, vitamins, or ribonucleotides. A novel well-designed scheme with integrated enzymatic conversions and fermentation enables the production of even complicated compounds, such as sugar nucleotides and oligosaccharides.

Inhibition of polyposis in the small intestine of BALB/c mice by intestinal bacteria.

The incidence of polyposis and the number of polyps per mouse were significantly lower in conventionalized (CVz) mice than in germ-free (GF) mice. There was no significant difference in the average number of polyps between GF and gnotobiotic (GB) mice monoassociated with the various strains of intestinal bacteria. However, the incidence of polyposis and the number of polyps per mouse were significantly lower for mice associated with either chloroform-resistant bacteria (CRB) or fusiform bacteria (FB) than for GF mice. This study demonstrated that polyposis was suppressed by FB and CRB in the small intestine of BALB/c mice.

Spontaneous polyposis in the small intestine of germ-free and conventionalized BALB/c mice.

The spontaneous polyposis in the small intestine of germfree (Gf) and conventionalized (Cv) BALB/c mice was studied. Gf mice were bred in our laboratory and maintained Gf in vinyl isolators. The first generation offspring of the Cv mice derived from the Gf mice was used as Cv animals. When they were 12 months old, the animals were killed under CO2 inhalation and autopsied carefully for the number and size of polyps with the aid of a dissecting microscope. The incidence of polyposis was higher in the Gf mice (68% in female and 89% in male) than in the Cv mice (37% in female and 51% in male). The number of polyps/mouse was also higher in the Gf mice (2.3 in female and 5.7 in male) than in the Cv mice (0.8 in female and 1.3 in male). All of the polyps were histopathologically adenomatous and developed only in the upper part (mainly duodenum) of the small intestine. The present study demonstrated that development of polyposis in the small intestine of BALB/c mice was suppressed by the presence of intestinal microflora.

In vivo augmentation of the cytotoxicity of spleen lymphocytes against syngeneic B-16 melanoma cells and the suppression of the artificial metastases in C57BL/6 mice by subcutaneous multiple injections of high dose human recombinant interleukin-2 (rIL-2).

The cytotoxicity of spleen lymphocytes against YAC-1 cells, against syngeneic B-16 and F-10 melanoma cells was augmented not only by incubation of spleen lymphocytes with human recombinant interleukin-2 (rIL-2) in vitro, but also by injecting C57BL/6 mice with high dose rIL-2 for more than 3 consecutive days. In animals injected s.c. with multiple high dose rIL-2, the numbers of tumor nodules in the lung were significantly decreased 21 days after i.v. tumor inoculation. In addition, in these groups of animals no liver metastases were observed although liver metastases were detected in 6/11 control mice.

Analysis of natural killer activity and antibody-dependent cellular cytotoxicity in healthy volunteers and in patients with primary lung cancer and metastatic pulmonary tumors.

To clarify the contribution of ADCC and NK activities to host immune response against cancer, the characteristics of cells mediating these activities were examined in the peripheral blood lymphocytes of normal volunteers, and the changes of these activities were also evaluated in patients with lung cancer and metastatic pulmonary tumors before and after chemotherapy. OAT cells derived from small cell carcinoma of the lung and K-562 cells derived from erythroleukemia were used as target cells of ADCC and/or NK assay. ADCC and NK activities were not changed according to age, sex, and blood type. Mild and marked personal difference were observed in ADCC and NK activity, respectively. These activities were also influenced by environment. ADCC and NK activities of normal adult volunteers were diversely correlated at the coefficient of gamma-0.426. NK activities were high against K-562 and CCRF-CEM cells, and low against BALL and OAT cells. NK activity against K-562 cells was strongly inhibited by K-562 or CCRF-CEM cells with high NK sensitivity, on the other hand, it was slightly inhibited by OAT and BALL cells with low NK sensitivity. NK activity against OAT cells was strongly inhibited by OAT, K-562 and CCRF-CEM cells, but not inhibited by BALL cells. The effector cells mediating NK activity were identified as non-adherent, E-receptor-positive, Fc-receptor-positive small lymphocytes. NK activity was not decreased before chemotherapy in patients with stage III primary lung cancer and metastatic pulmonary tumors. It was decreased only in patients of bad performance status, and it was significantly decreased in all patients after chemotherapy. ADCC also exhibited the tendency to decrease after chemotherapy in tumor-bearing patients. The recovery of NK-activity after chemotherapy well correlated with the effect of chemotherapy.

Analysis of metastatic spread and growth of tumor cells in mice with depressed natural killer activity by anti-asialo GM1 antibody or anticancer agents.

The mechanism of artificial and spontaneous metastases of tumor was analyzed in B16 melanoma cells and C57BL/6 mice by using anti-asialo GM1 antibody and anticancer agents. Single administrations of 500 micrograms anti-asialo GM1 antibody resulted in significantly decreased NK activity in spleen cells of C57BL/6 mice, lasting 10 days from the day following administration. Treatment with anti-asialo GM1 antibody never decreased the function of T lymphocytes measured by blastogenesis with phytohemagglutinin or T cell growth factor. The tumoricidal functions of activated macrophages but not of resident macrophages were decreased by in vivo treatment with anti-asialo GM1 antibody. The anti-asialo GM1 antibody was evaluated in terms of the enhancing effect on pulmonary metastases with regard to the timing of administration. Treatment with anti-asialo GM1 antibody 1 day before or on the day of tumor inoculation resulted in a substantial increase in the number of artificial pulmonary metastases. In the experimental system of spontaneous metastases, anti-asialo GM1 antibody most effectively increased the number of pulmonary metastases when administered 1-2 weeks before the removal of primary tumor, when the tumor cells are thought to be released into blood circulation from the primary site. In addition, accelerated growth of transplanted tumors at the primary site was observed in mice treated with anti-asialo GM1 antibody. These results strongly suggest that anti-asialo GM1 antibody enhances the incidence of in vivo tumor metastases and the growth of transplanted tumor mainly by suppressing the function of NK cells. The maximum effective dose (MED) of mitomycin C or its derivative (M-83) suppressed NK activity significantly, and pretreatment with these anticancer agents enhanced the growth of the artificial pulmonary and liver metastases. In contrast, the MED of cDDP showed no effect on the NK activity or the numbers of pulmonary and liver metastases. These results indicate that the depression of NK activity induced by chemotherapy results in the promotion of metastatic disease. From these studies it can be concluded that NK cells have a key role in the control of metastases of malignant disease, and that support of NK activity is very important for the prevention of metastases.

A cold-adapted protease engineered by experimental evolution system.

A new cold-adapted protease subtilisin BPN' mutant, termed m-51, was successfully isolated by use of an evolutionary program consisting of two-step in vitro random mutagenesis, which we developed for the screening of mutant subtilisins with increased activity at low temperature. The m-51 mutant showed 70% higher catalytic efficiency, expressed by the k(cat)/K(m) value, than the wild-type at 10 degrees C against N-succinyl-L-Ala-L-Ala-L-Pro-L-Phe-p-nitroanilide as a synthetic substrate. This cold-adaptation was achieved mainly by the increase in the k(cat) value in a temperature-dependent manner. Genetic analysis revealed that m-51 had three mutations, Ala-->Thr at position -31 (A-31T) in the prodomain, Ala-->Val at position 88 (A88V), and Ala-->Thr at position 98 (A98T). From kinetic parameters of the purified mutant enzymes, it was found that the A98T mutation led to 30% activity increase, which was enhanced up to 70% by the accompanying neutral mutation A88V. The A-31T mutation severely constrained the autoprocessing-mediated maturation of the pro-subtilisin in the Escherichia coli expression system, thus probably causing an activity-non-detectable mutation in the first step of mutagenesis. No distinct change was observed in the thermal stability of any mutant or in the substrate specificity for m-51. In the molecular models of the two single mutants (A88V and A98T), relatively large displacements of alpha carbon atoms were found around the mutation points. In the model of the double mutant (A88V/A98T), on the other hand, the structural changes around the mutation point counterbalanced each other, and thus no crucial displacements occurred. This mutual effect may be related to the enhanced activity of the double mutant.

Injection sclerotherapy of esophageal varices for patients undergoing emergency and elective surgery.

From October 1977 to September 1981, 68 patients with esophageal varices (30 emergency cases of bleeding and 38 elective cases) were treated by injecting 5% ethanolamine oleate into varices, using an esophagofiberscope. Esophageal bleeding was successfully controlled in 29 of 30 patients who had emergency surgery. None of the 38 patients who underwent elective operation had bleeding after treatment. When recurrence occurred 1 or 2 years after treatment, the same procedure was repeated. Pleuritis occurred in one of the patients who had emergency surgery, and bleeding (300 to 400 ml) from the esophagocardial junction occurred in two patients who underwent elective operation. These patients were treatment conservatively.