Biocatalytic synthesis of flavones and hydroxyl-small molecules by recombinant Escherichia coli cells expressing the cyanobacterial CYP110E1 gene

Background: Cyanobacteria possess several cytochrome P450s, but very little is known about their catalytic functions. CYP110 genes unique to cyanaobacteria are widely distributed in heterocyst-forming cyanobacteria including nitrogen-fixing genera Nostoc and Anabaena. We screened the biocatalytic functions of all P450s from three cyanobacterial strains of genus Nostoc or Anabaena using a series of small molecules that contain flavonoids, sesquiterpenes, low-molecular-weight drugs, and other aromatic compounds. Results: Escherichia coli cells carrying each P450 gene that was inserted into the pRED vector, containing the RhFRed reductase domain sequence from Rhodococcus sp. NCIMB 9784 P450RhF (CYP116B2), were co-cultured with substrates and products were identified when bioconversion reactions proceeded. Consequently, CYP110E1 of Nostoc sp. strain PCC 7120, located in close proximity to the first branch point in the phylogenetic tree of the CYP110 family, was found to be promiscuous for the substrate range mediating the biotransformation of various small molecules. Naringenin and (hydroxyl) flavanones were respectively converted to apigenin and (hydroxyl) flavones, by functioning as a flavone synthase. Such an activity is reported for the first time in prokaryotic P450s. Additionally, CYP110E1 biotransformed the notable sesquiterpene zerumbone, anti-inflammatory drugs ibuprofen and flurbiprofen (methylester forms), and some aryl compounds such as 1-methoxy and 1-ethoxy naphthalene to produce hydroxylated compounds that are difficult to synthesize chemically, including novel compounds. Conclusion: We elucidated that the CYP110E1 gene, C-terminally fused to the P450RhF RhFRed reductase domain sequence, is functionally expressed in E. coli to synthesize a robust monooxygenase, which shows promiscuous substrate specificity (affinity) for various small molecules, allowing the biosynthesis of not only flavones (from flavanones) but also a variety of hydroxyl-small molecules that may span pharmaceutical and nutraceutical industries

Mixed Type of Malignant Mesothelioma in an Aged Male ICR Mouse

Multiple whitish nodules in the thoracic cavity at the site of the thymus were observed in a 101-week-old male ICR mouse. In a histopathological examination, the neoplastic cells were predominantly fusiform in shape and proliferated in sarcomatoid growth patterns. Some neoplastic cells showed epithelial growth patterns, such as the ductal structures. Mitotic figures were frequently seen, and small necrotic foci and invasion to adjacent thoracic organs were noted. In Alcian blue staining, bluish materials were observed between fusiform-shaped cells and in some of the lumens of the ductal structures. In immunohistochemistry, both fusiform-shaped and ductal structure-forming cells were positive for vimentin and weakly positive to positive for cytokeratin. Based on the aforementioned findings, the thoracic nodules were diagnosed as a mixed type of malignant mesothelioma. This case was thought to be rare because of the very low occurrence of spontaneous mesothelioma in mice

Epidemiological Situation of Dengue Infection in Lao P. D. R.

In 1979, 37 cases of dengue hemorrhagic fever (DHF) were reported to the Ministry of Health for the first time in Laos. In 1985, a total of 1,759 cases of DHF was reported as the first epidemic. In 1987, larger epidemics of DHF occurred with a total of 6,567 cases in the Vientiane Municipality and a total of 3,098 cases in four provinces. The morbidity rate of the outbreak in Vientiane was as high as 1,530/100,000. In the end of 1988, an Aedes Control Unit (ACU) was set up in the Vientiane Municipality to reduce the Aedes larva density and strengthen health education to the residents. Since 1989, DHF epidemic such as seen in 1985 or 1987 has not occurred in Vientiane so far. Activities of the ACU seem to be efficacious in reducing the larva density, judged by the Breteau and House indices obtained. However, another approach to show the reduction of dengue virus transmission has not been tried yet. In 1990, a seroepidemiological study on inhabitants in the Vientiane Municipality was performed. The results indicated followings: (1) The percentage prevalences of neutralizing (N) antibodies to dengue (DEN) 1-4 viruses increased with age, but the prevalences of N antibodies to DEN 3 and DEN 4 were lower than those to DEN 1 and DEN 2 viruses. (2) The geometric mean titer of N antibody to DEN 2 was highest among 4 serotypes in every age group. (3) By the age of 15 years, the majority (90%) of the inhabitants were infected with two or more serotypes of DEN virus

Arbovirus Infections in Pilot Areas in Laos

Since 1993, virological and seroepidemiological survey of arbovirus infections was conducted in pilot areas in Laos under the World Health Organization-Japan International Cooperation Agency-Laos trilateral Primary Health Care Project. Human sera were obtained at the laboratory in Khammouane Provincial Hospital, and at Sok Yai village in Vientiane Municipality, and antibodies to Japanese encephalitis (JE), dengue (DEN) and chikungunya (CHIK) viruses were assayed by neutralization test. The sera were classified according to the age groups. In Khammouane area, antibody-positive rates to DEN-1, 2, and 3 reached high (over 90%) by 11-15 years old and kept high thereafter. Antibody-positive rates to DEN-4 and JE increased with age and reached over 50% by 21-30 and 31-40 years old, respectively. In Sok Yai area, antibody-positive rates to DEN-2 and 3 were over 50% in all age groups. The positive rate of DEN-1 antibody reached 50% by 11-15 years old and increased with age. Although DEN-4 and JE antibody-positive rates fluctuated among age groups, they tended to increase with age. JE antibody survey in swine sera indicated that the JE virus was active during the rainy season. Positive rate of CHIK antibody tended to increase with age and reached over 50% in older age groups. These results indicate that DEN-1, 2, and 3 viruses are circulating more frequently in these areas. DEN-4, JE and CHIK viruses also exist with less activity. In terms of the primary health care for the prevention and control of arbovirus infections, the project team conducted health education and environmental sanitation in Sok Yai village. They also organized several training courses on the diagnosis and treatment of JE and DEN for junior doctors, health workers, and laboratory technicians

Atrasentan and renal events in patients with type 2 diabetes and chronic kidney disease (SONAR): a double-blind, randomised, placebo-controlled trial

Background: Short-term treatment for people with type 2 diabetes using a low dose of the selective endothelin A receptor antagonist atrasentan reduces albuminuria without causing significant sodium retention. We report the long-term effects of treatment with atrasentan on major renal outcomes. Methods: We did this double-blind, randomised, placebo-controlled trial at 689 sites in 41 countries. We enrolled adults aged 18–85 years with type 2 diabetes, estimated glomerular filtration rate (eGFR)25–75 mL/min per 1·73 m 2 of body surface area, and a urine albumin-to-creatinine ratio (UACR)of 300–5000 mg/g who had received maximum labelled or tolerated renin–angiotensin system inhibition for at least 4 weeks. Participants were given atrasentan 0·75 mg orally daily during an enrichment period before random group assignment. Those with a UACR decrease of at least 30% with no substantial fluid retention during the enrichment period (responders)were included in the double-blind treatment period. Responders were randomly assigned to receive either atrasentan 0·75 mg orally daily or placebo. All patients and investigators were masked to treatment assignment. The primary endpoint was a composite of doubling of serum creatinine (sustained for ≥30 days)or end-stage kidney disease (eGFR <15 mL/min per 1·73 m 2 sustained for ≥90 days, chronic dialysis for ≥90 days, kidney transplantation, or death from kidney failure)in the intention-to-treat population of all responders. Safety was assessed in all patients who received at least one dose of their assigned study treatment. The study is registered with ClinicalTrials.gov, number NCT01858532. Findings: Between May 17, 2013, and July 13, 2017, 11 087 patients were screened; 5117 entered the enrichment period, and 4711 completed the enrichment period. Of these, 2648 patients were responders and were randomly assigned to the atrasentan group (n=1325)or placebo group (n=1323). Median follow-up was 2·2 years (IQR 1·4–2·9). 79 (6·0%)of 1325 patients in the atrasentan group and 105 (7·9%)of 1323 in the placebo group had a primary composite renal endpoint event (hazard ratio [HR]0·65 [95% CI 0·49–0·88]; p=0·0047). Fluid retention and anaemia adverse events, which have been previously attributed to endothelin receptor antagonists, were more frequent in the atrasentan group than in the placebo group. Hospital admission for heart failure occurred in 47 (3·5%)of 1325 patients in the atrasentan group and 34 (2·6%)of 1323 patients in the placebo group (HR 1·33 [95% CI 0·85–2·07]; p=0·208). 58 (4·4%)patients in the atrasentan group and 52 (3·9%)in the placebo group died (HR 1·09 [95% CI 0·75–1·59]; p=0·65). Interpretation: Atrasentan reduced the risk of renal events in patients with diabetes and chronic kidney disease who were selected to optimise efficacy and safety. These data support a potential role for selective endothelin receptor antagonists in protecting renal function in patients with type 2 diabetes at high risk of developing end-stage kidney disease. Funding: AbbVie