Incidence and risk factors for norovirus-related diarrhea in Japanese geriatric intermediate care facilities: A prospective cohort study.

AIM: The risk of developing infectious diarrhea among elderly residents at Japanese geriatric intermediate care facilities is unclear. We investigated the incidence rate and risk factors of norovirus-related diarrhea at such facilities. METHODS: This prospective cohort study followed 1727 residents from November 2018 to April 2020 at 10 geriatric intermediate care facilities in Osaka, Japan regarding the occurrence of diarrhea. Resident data were collected from their medical records using structured forms at two to three of the following three time points: at recruitment, if they developed diarrhea, and when they left the facility. Residents who developed diarrhea were tested using rapid diagnostic tests for norovirus. Cox proportional hazard model was employed to hazard ratios (HRs) with 95% confidence intervals (CIs) to estimate the risk factors for norovirus-related diarrhea. RESULTS: During the study period, 74 residents developed diarrhea, 13 of whom were norovirus positive. The incidence rate of norovirus-related diarrhea was 10.11 per 1000 person-years (95% CI: 4.61-15.61). In terms of risk factors, people with care-needs level 3 were at a higher risk for developing norovirus-related diarrhea (adjusted HR [aHR] = 7.35, 95% CI: 1.45-37.30). Residents with hypertension (aHR = 3.41, 95% CI: 1.05-11.04) or stroke (aHR = 8.84, 95% CI: 2.46-31.83), and those who walked with canes (aHR = 16.68, 95% CI: 1.35-206.52) also had a significantly higher risk for norovirus-related diarrhea. CONCLUSIONS: Throughout the study period, the incidence of development of diarrhea was low. Care-needs level 3, stroke, hypertension and use of a cane were identified as risk factors for norovirus-related diarrhea in Japanese geriatric intermediate care facilities. Geriatr Gerontol Int 2023; 23: 179-187.

Effects of heat treatment on conformation and cell growth activity of alpha- lactalbumin and beta-lactoglobulin from market milk.

Heat processes, low temperature for long time (LTLT) pasteurization and ultra-heat treatment (UHT) sterilization, are essential for commercial market milk to improve the shelf life of raw milk and ensure microbial safety. We evaluated the effects of heat experience on the molecular properties of α-lactalbumin (α-LA) and ß-lactoglobulin (ß-LG) isolated from four types of market milk such as LTLT-A (66°C for 30 min), LTLT-B (65°C for 30 min), UHT-I (130°C for 2 s, indirect heating) and UHT-D (135°C for 2 s, direct heating) samples. We examined molecular conformations using circular dichroism spectrum measurement and cell growth activity using the WST-1 method for the proteins. α-LA isolated from each of these four types of market milk displayed no significant structural difference as compared to raw milk α-LA, while α-LA of UHT-I only inhibited cell growth of an intestinal epithelial cell line more potently than raw milk α-LA. In the case of ß-LG, only the UHT-I sample demonstrated a drastic change in structure, while it did not exhibit any cytotoxicity. We found that cell viability effects of α-LA and ß-LG are attributable to the type of UHT; indirect and direct. These findings indicate that the effect of heat treatment on whey proteins should carefully be investigated further.

Priorities of health care outcomes for the elderly.

OBJECTIVES: Physicians are uncertain about what medical services should be provided to older and/or disabled patients. Better understanding of health outcome prioritization among health care providers and recipients may help the process of decision- and policy-making. For this purpose, surveys were conducted on priorities of health care outcomes for the elderly. DESIGN: Survey research. SETTING: Four groups of health care providers and four groups of health care recipients. PARTICIPANTS: A total of 2512 health care providers and 4277 recipients. MEASUREMENTS: Questionnaires were sent to more than 8000 health care providers and more than 9000 health care recipients: geriatricians, physicians who commonly see older patients or work in long term care facilities, staff members and participants in adult day care, patients in outpatient geriatric clinics, family members of patients with dementia, and community-dwelling older adults. The questionnaire asked the subjects to rank 12 measures of health care outcomes. RESULTS: The mean response rate was 49%. All health care provider groups considered "improvement of quality of life" the most important. In contrast, in health care recipient groups, "effective treatment of illness," "improvement of physical function," and "reduction of carer burden" were given high priority, whereas "improvement of quality of life" was perceived as less important. All the groups, including health care providers and recipients, ranked "reduction of mortality" the least important, followed by "avoiding institutional care." Stratification analysis showed that the results did not differ by sex, nursing care level, or the existence of relatives who required nursing care, whereas age slightly influenced the order of high-ranked measures. CONCLUSION: Priorities of health care services and their differences between providers and recipients should be taken into account in the health care of older patients and the design of health care policies and research.

Hyperproduction of sebaceous cis-6-hexadecenoic acid by esterase-reduced mutant of Rhodococcus sp. strain.

cis-6-Hexadecenoic acid is a major component of human sebaceous lipids that is involved in skin self-sterilization and atopic dermatitis amelioration. It can be prepared by hydrolysis of isopropyl cis-6-hexadecenoate produced by resting cells of Rhodococcus sp. strain KSM-MT66. To devise an economical industrial-scale process for the production of this rare fatty acid, we optimized the conditions for growing rhodococcal cells. Mg(2+) and Fe(2+) ions are essential for the efficient production of isopropyl cis-6-hexadecenoate. To further increase the production of isopropyl cis-6-hexadecenoate, we created a mutant strain (T64) with reduced esterase activity by random mutagenesis using UV irradiation of MT66. Under an optimized condition, the mutant T64 produced more than 60 g l(-1) isopropyl cis-6-hexadecenoate in a 4-d cultivation, corresponding to about 52 g l(-1)cis-6-hexadecenoate.

Intragenomic diversity of the V1 regions of 16S rRNA genes in high-alkaline protease-producing Bacillus clausii spp.

Alkaliphilic Bacillus sp. strain KSM-K16, which produces high-alkaline M-protease, was characterized phenotypically, biochemically and genetically. This strain was identified as Bacillus clausii based on the results of taxonomic studies, including sequencing of the 16S rRNA gene and DNA-DNA hybridization. Seven rRNA operons in the genome were identified by pulsed-field gel electrophoresis. Sequencing of cloned 16S rRNA genes revealed two distinct types of variable region V1. Moreover, some cloned 16S rRNA genes in some of the reference strains of B. clausii had a V1 region of yet another type. The B. clausii strains could clearly be divided into at least two subgroups based on the frequencies of the types of cloned V1 sequence. Bacillus sp. strain KSM-K16 was found to be in a different phylogenetic position from other high-alkaline protease-producing strains of B. clausii.

Endoglucanases from Paenibacillus spp. form a new clan in glycoside hydrolase family 5.

Endoglucanase (Egl)-producing bacteria from soil samples were screened using insoluble cellulosic substrates as sole carbon sources at alkaline pH (pH 9-10). Four Egls with Avicelase activity at alkaline pH were found in the culture broth of each isolate. The Egl genes of the isolates (all Paenibacillus spp.) were shotgun cloned and sequenced-all had a 1752bp open reading frame (584 amino acids) with a putative signal sequence (33 amino acids), and encoded mature enzymes of 551 amino acids (58,360-58,672Da). The mature enzymes showed a high degree of similarity to each other (>93% identity), with the next closest similarity to Egl3a of a patented strain of Paenibacillus lautus NCIMB 40250 (81.5-87.3% identity). These enzymes showed low similarity to other known Egls with less than 50% identity. A representative recombinant enzyme degraded lichenan, carboxymethylcellulose (CMC), glucomannan, acid or alkaline swollen celluloses, and microcrystalline cellulose (Avicel). The optimal pH and temperature of the recombinant enzyme for degrading CMC and Avicel were pH 6.0-8.5 and 45-55 degrees C, respectively. Egls belong to glycoside hydrolase family 5 and form a distinct clan based on the phylogenetic analysis of their amino acid sequences.

Extracellular production of human cystatin S and cystatin SA by Bacillus subtilis.

We herein describe the development of a Bacillus subtilis system that can be used to produce large quantities of recombinant (r-) human salivary cystatins, a cysteine protease inhibitor of family 2 in the cystatin superfamily. The B. subtilis that lacked the alkaline protease E gene (DeltaaprE type mutant strain) was prepared by homologous recombination. The cDNA fragments coding for mature cystatins (S and SA) were ligated in frame to the DNA segment for the signal peptide of endoglucanase in the pHSP-US plasmid vector that was then use to transform the DeltaaprE type mutant strain of B. subtilis. The transformants carrying the expression vectors were cultivated in 5-L jar fermenters for 3 days at 30 degrees C. Both r-cystatin S and r-cystatin SA were successfully expressed and secreted into the culture broth, and were purified using a fast performance liquid chromatography system. The first use of DeltaaprE type mutant strain of B. subtilis made it possible to obtain a high yield of secreted protein, which makes this system an improvement over expression in Escherichia coli. We conclude that this system has high utility for expression of commercial quantities of secreted proteins.

A new subtilisin family: nucleotide and deduced amino acid sequences of new high-molecular-mass alkaline proteases from Bacillus spp.

Six genes encoding high-molecular-mass subtilisins (HMSs) of alkaliphilic Bacillus spp. were cloned and sequenced. Their open reading frames of 2,394-2,424 bp encoded prosubtilisins of 798-808 amino acids (aa) consisting of the prepropeptides of 151-158 aa and the mature enzymes of 640-656 aa. The deduced aa sequences of the mature enzymes exhibited 60-95% identity to those of FT protease of Bacillus sp. strain KSM-KP43, a subtilisin-like serine protease, and a minor serine protease, Vpr, of Bacillus strains. Three of the six recombinant enzymes were susceptible to proteolysis, but the others were autodigestion resistant. All enzymes had optimal pH values of 10.5-11.0, optimal temperatures of 40-45 degrees C for hydrolysis of a synthetic substrate, and were heat labile. These alkaline proteases seem to form a new subtilisin family, as judged by their aa sequences and phylogenetic analysis.

Nucleotide and deduced amino acid sequences of a high-molecular-mass subtilisin from an alkaliphilic Bacillus isolate.

A high-molecular-mass subtilisin was found in culture broth of the alkaliphilic Bacillus sp. strain KSM-KP43. The gene encoding the enzyme (FT protease) was determined using a mixed primer designed from the N-terminal amino acid (aa) sequence of the purified enzyme. The determined nucleotide sequence of the gene consisted of a 2427-bp open reading frame (ORF) that encoded a putative prepro-peptide (152 aa) and a mature enzyme (656 aa; 68,506 Da). The deduced aa of the mature enzyme revealed a moderate homology to a subtilisin-type proteinase from Bacillus halodurans and a minor extracellular protease, Vpr, from Bacillus subtilis with 64% and 57% identity, respectively. The molecular mass of the purified recombinant FT protease was approximately 72 kDa as judged by both SDS-polyacrylamide gel electrophoresis (PAGE) and gel filtration. FT protease showed maximal activity toward glutaryl-Ala-Ala-Pro-Leu-p-nitroanilide at pH 10.5 and at 45 degrees C. The enzyme was rapidly inactivated by incubation over 45 degrees C for 15 min at both pH 7 and 10. Calcium ions were slightly protective for thermoinactivation of the enzyme.

Nucleotide and deduced amino acid sequences of a new subtilisin from an alkaliphilic Bacillus isolate.

The gene for a new subtilisin from the alkaliphilic Bacillus sp. KSM-LD1 was cloned and sequenced. The open reading frame of the gene encoded a 97 amino-acid prepro-peptide plus a 307 amino-acid mature enzyme that contained a possible catalytic triad of residues, Asp32, His66, and Ser224. The deduced amino acid sequence of the mature enzyme (LD1) showed approximately 65% identity to those of subtilisins SprC and SprD from alkaliphilic Bacillus sp. LG12. The amino acid sequence identities of LD1 to those of previously reported true subtilisins and high-alkaline proteases were below 60%. LD1 was characteristically stable during incubation with surfactants and chemical oxidants. Interestingly, an oxidizable Met residue is located next to the catalytic Ser224 of the enzyme as in the cases of the oxidation-susceptible subtilisins reported to date.

Vinculin: a novel marker for quiescent and activated hepatic stellate cells in human and rat livers.

In liver injuries, the quiescent hepatic stellate cells (HSCs) promptly change to activated HSCs, which are easily identified by the intense immunoreactivity for alpha-smooth muscle actin. However, reproducible markers for quiescent HSCs in formalin-fixed, paraffin-embedded liver tissue sections have not yet been reported. We immunohistochemically examined the expression of vinculin, one major protein of dense plaques, on cultured LI90 cells and on HSCs in normal and diseased human and rat livers. In cultured LI90 cells, vinculin appeared as small linear patches. Although vinculin was consistently negative in the routine liver tissue sections, an antigen retrieval technique using microwave oven heating yielded excellent effects. Using this technique, the formalin-fixed, paraffin-embedded human and rat normal liver tissue sections showed the vinculin immunoreactivity along the sinusoidal wall. Immunoelectron microscopic observation of hepatic parenchyma demonstrated that the vinculin was exclusively expressed in quiescent HSCs. In fetal rat livers, vinculin-positive quiescent HSCs gradually increased in number with gestation. In diseased livers the activated HSCs showed more intense immunoreaction for vinculin. These results indicate that, using microwave pretreatment, vinculin is expressed in quiescent and activated HSCs in routinely processed liver tissue sections. It could allow us to evaluate the development and distribution of quiescent HSCs and to examine the relationship between quiescent and activated HSCs.

Early gastric cancer giving rise to bone and brain metastases--a review of the Japanese literature.

A forty-year-old female consulted a hospital complaining of epigastralgia. She underwent endoscopy, which showed irregular shaped ulceration with fold convergence; the biopsy specimen revealed poorly differentiated adenocarcinoma. She underwent subtotal gastrectomy and lymph node dissection. Histological findings revealed the signet-ring-cell cancer confined to the mucosa and no lymph node metastasis. The serum carcinoembryonic antigen was elevated 2 years and 11 months after operation. Bone scintigraphy demonstrated multiple accumulation and bone biopsy of the sacrum revealed the metastatic gastric cancer. She underwent chemotherapy and radiation, however, later complained of nausea, vomiting, and diminished visual acuity. Brain computed tomography revealed multiple brain metastasis. She died 3 years and 6 months after her operation. We reviewed the 39 reported cases of early gastric cancer with bone metastasis in Japan, which suggests that signet-ring-cell carcinoma and poorly differentiated carcinoma have a possibility of bone metastasis even though the early gastric cancer is confined to the mucosa.

A novel species of alkaliphilic Bacillus that produces an oxidatively stable alkaline serine protease.

A novel gram-positive, strictly aerobic, motile, sporulating, and facultatively alkaliphilic bacterium designated KSM-KP43 was isolated from a sample of soil. The results of 16S rRNA sequence analysis placed this bacterium in a cluster with Bacillus halmapalus. However, the level of the DNA-DNA hybridization of KSM-KP43 with B. halmapalus was less than 25%. Moreover, the G + C contents of the genomic DNA were 41.6 mol% for KSM-KP43 and 38.6 mol% for B. halmapalus. Because there were also differences in physiological properties and cellular fatty acid composition between the two organisms, we propose KSM-KP43 as a novel species of alkaliphilic Bacillus. This novel strain produces a new class of protease, an oxidatively stable serine protease that is suitable for use in bleach-based detergents. The enzyme contained 640 amino acid residues, including a possible approximately 200-amino-acid prepropeptide in the N-terminal and a unique stretch of approximately 160 amino acids in the C-terminal regions (434-amino-acid mature enzyme with a calculated molecular mass of 45,301 Da). The C-terminal half after the putative catalytic Ser255 and the contiguous C-terminal extension shared local similarity to internal segments of a membrane-associated serine protease of a marine microbial assemblage and the serine protease/ABC transporter precursors of the slime mold Dictyostelium discoideum, and to the C-terminal half of a cold-active alkaline serine protease of a psychrotrophic Shewanella strain.