Design of a Novel Antimicrobial Peptide Activated by Virulent Proteases

Antimicrobial peptides are promising antibiotics as they possess strong antimicrobial activity and very broad spectra of activity. However, administration of an antibiotic with a very broad spectrum of activity disrupts normal microflora and increases the risks of other fatal infections. To solve the problem, we designed a novel antimicrobial peptide that is activated by virulent proteases of pathogenic organisms. We constructed a peptide composed of three domains, namely an antimicrobial peptide (lactoferricin) as the active center, a protective peptide (magainin intervening sequence) that suppresses antimicrobial activity, and a specific linker that joins these two components and is efficiently cleaved by virulent proteases. We utilized Candida albicans as a model organism that produces secreted aspartic proteases as a virulence attribute. We screened for a peptide sequence efficiently cleaved by secreted aspartic proteases isozymes and identified a GFIKAFPK peptide as the most favorable substrate. Subsequently, we chemically synthesized a peptide containing the GFIKAFPK sequence. The designed peptide possessed no antimicrobial activity until it was activated by secreted aspartic proteases isozymes. Furthermore, it demonstrated selective antimicrobial activity against C. albicans, but not against Saccharomyces cerevisiae. A designed peptide like the one described in this study may protect normal microflora, resulting in enhanced safety as a therapeutic.     

Duodenal stenosis caused by cystic dystrophy in heterotopic pancreas: Report of a case

We herein describe the first reported case of duodenal stenosis caused by cystic dystrophy in heterotopic pancreas (CDHP) in Asia. A 63-year-old man was admitted to the hospital presenting with nausea and vomiting of 2 days’ duration. Laboratory examinations showed an elevation in both the serum amylase level (275 IU/l) and white blood cell count (13 600/μl). A 3-cm-diameter tumor close against the duodenum was pointed out from the results of computed tomography (CT) and magnetic resonance cholangiopancreatography (MRCP); the tumor contained a cystic and solid component. Endoscopic examinations and an upper gastrointestinal series showed stenosis of the second portion of the duodenum without any mucosal change. The tumor was considered to be located at the submucosal layer of the second duodenum. The biopsy specimen of the duodenum revealed no malignancy. We strongly doubted the presence of a malignant submucosal tumor in the duodenum based on the findings of diagnostic imaging, and a pancreaticoduodenectomy was thus performed. Histopathologically, the lesion was diagnosed to be CDHP. The postoperative course was uneventful. The patient was symptomatic but was free of any symptoms after surgery. He continues to be regularly followed up on an outpatient basis and has had no recurrence of symptoms. This case demonstrates the need to consider CDHP in the differential diagnosis as a rare cause of duodenal stenosis.     

Anaerobic NADH-fumarate reductase system is predominant in the respiratory chain of Echinococcus multilocularis, providing a novel target for the chemotherapy of alveolar echinococcosis

Alveolar echinococcosis, which is due to the massive growth of larval Echinococcus multilocularis, is a life-threatening parasitic zoonosis distributed widely across the northern hemisphere. Commercially available chemotherapeutic compounds have parasitostatic but not parasitocidal effects. Parasitic organisms use various energy metabolic pathways that differ greatly from those of their hosts and therefore could be promising targets for chemotherapy. The aim of this study was to characterize the mitochondrial respiratory chain of E. multilocularis, with the eventual goal of developing novel antiechinococcal compounds. Enzymatic analyses using enriched mitochondrial fractions from E. multilocularis protoscoleces revealed that the mitochondria exhibited NADH-fumarate reductase activity as the predominant enzyme activity, suggesting that the mitochondrial respiratory system of the parasite is highly adapted to anaerobic environments. High-performance liquid chromatography-mass spectrometry revealed that the primary quinone of the parasite mitochondria was rhodoquinone-10, which is commonly used as an electron mediator in anaerobic respiration by the NADH-fumarate reductase system of other eukaryotes. This also suggests that the mitochondria of E. multilocularis protoscoleces possess an anaerobic respiratory chain in which complex II of the parasite functions as a rhodoquinol-fumarate reductase. Furthermore, in vitro treatment assays using respiratory chain inhibitors against the NADH-quinone reductase activity of mitochondrial complex I demonstrated that they had a potent ability to kill protoscoleces. These results suggest that the mitochondrial respiratory chain of the parasite is a promising target for chemotherapy of alveolar echinococcosis.     

Size of the perivitelline space and incidence of polyspermy in rabbit and hamster oocytes

Purpose  

The size of the perivitelline space and the incidence of polyspermy were observed in ovulated and cultured oocytes from rabbits and hamsters with or without treatment by 4-methylumbelliferone (MU), an inhibitor of hyaluronic acid (HA) synthase, in order to examine the relationship between the incidence of polyspermy and the size of the perivitelline space. The amount of HA in the medium with MU-treated hamster oocytes was measured and compared with that in the medium with untreated oocytes.     

Questionnaire survey on sleep habit of 3-year-old children in Asahikawa City

The present study surveyed the sleep habits of 3-year-old children in Asahikawa city using questionnaires completed by a parent during children's medical check-ups. Questionnaires were collected from the parents of 404 children (209 males, 195 females; mean age, 3.1 years) enrolled in this survey. Among these children, the mean bedtime was 9.6 PM with 145 children (36%) going to bed after 10 PM. On the other hand, the mean wake-up time was 7.5 AM, with 123 children (30%) waking up after 8 AM. The mean nocturnal sleep duration was 10.1 hours. Nocturnal sleep durations in children that went to bed after 10 PM were significantly shorter than in children who went to bed earlier (p < 0.01). Seventy-three percent of the children had a daily afternoon nap. Twelve percent of these children usually awoke from their nap after 5 PM, and the mean bedtime for these children was 10.1 PM. A late bedtime was significantly correlated with parental complaints such as short-temper and poor appetite (p < 0.05). Although parents were concerned about night-time sleep conditions, they were not concerned about the daytime conditions which regulate children's sleep-wake rhythm, such as daylight exposure, daytime activity, and naps. While 24% of parents had complains regarding their children's sleep, only 3% had consulted with a doctor. Similar to the previous reports, the present findings demonstrate that children in Asahikawa city go to bed late and have decreased sleep duration. Since the establishment of a normal sleep-wake rhythm is essential for both physical and mental development in children, it is necessary to educate parents regarding the importance of children's sleep.     

Clonal change of methicillin‐resistant Staphylococcus aureus isolated from patients with impetigo in Kagawa,Japan

Recently, the USA300 clone, which is a Panton–Valentine leukocidin (PVL)‐positive clonal complex 8‐staphylococcal cassette chromosome mec type IV (CC8‐IV) community‐acquired methicillin‐resistant Staphylococcus aureus (CA‐MRSA) strain, emerged in community and hospital settings in Japan. Hence, clonal types of CA‐MRSA strains are predicted to be changing. Nonetheless, long‐term surveillance of CA‐MRSA has not been conducted in Japan. Here, we investigated the transition and current status of CA‐MRSA strains isolated from outpatients with impetigo; the samples were collected between 2007 and 2016 in Kagawa, Japan. The detection rate (22.8%, 488/2139 strains) of MRSA slightly decreased in these 10 years. Molecular epidemiological analyses showed that the prevalence of the CC89‐II clone, which is a typical CA‐MRSA genotype of causative agents of impetigo, significantly decreased from 48.0% (48/100 strains) in 2007–2009 to 21.9% (16/73 strains) in 2013–2016. By contrast, a non‐USA300 CC8‐IV clone, which is a highly pathogenic CA‐MRSA/J clone, significantly increased in prevalence from 9.0% (9/100 strains) to 32.9% (24/73 strains). The prevalence of PVL‐positive CA‐MRSA strains increased annually from 2012 (0%) to 2015 (6.7%), whereas only one of these strains turned out to be the USA300 clone. Antibiotic susceptibility data revealed that the rates of resistance to gentamicin and clindamycin among CA‐MRSA strains decreased along with the decreased prevalence of the CC89‐II clone and increased prevalence of the CA‐MRSA/J clone. Our data strongly suggest that the clonal types and antibiotic susceptibility of CA‐MRSA isolated from patients with impetigo dramatically changed during the last 10 years in Japan.