Identification of a novel splice variant of neuronal nitric oxide synthase, nNOSß, in myofilament fraction of murine cardiomyocytes.

Splice variant forms of neuronal nitric oxide synthase (nNOS or NOS1), nNOSα and nNOSµ, are well established to be functionally expressed in discrete compartments in cardiomyocytes (e.g. sarcoplasmic reticulum, SR, caveolae in plasma membrane or mitochondria). So far, whether nNOS is expressed in myofilament fraction of cardiomyocytes and the splice variant form of nNOS are unknown. Immunoblotting results using two nNOS specific antibodies (BD Transduction Laboratories aa 1095-1289 and Santa Cruz Biotechnology aa 2-300) clearly demonstrated that nNOS was abundantly expressed in myofilament-enriched fraction of cardiomyocytes. Whilst the molecular weight of nNOS in membrane/cytosol fractions was ∼165 kDa, nNOS in myofilament was below 140 kDa, suggesting that the predominant splice variant of nNOS in myofilament is nNOSß. RT-PCR results confirmed the expressions of both nNOSα and nNOSß mRNAs in rat cardiomyocytes. Similarly, immunoprecipitation experiments using myofilament lysates of cardiomyocytes identified nNOS with low molecular weight (M.W. ∼140 kDa), confirming nNOSß. Intriguingly, all three splice variants of nNOS were undetectable in the lysates of cardiomyocytes (including myofilament fractions) from nNOS-/- mice (which lacks nNOSα/µ). Furthermore, nNOSß expression in myofilament of cardiomyocytes was not different in hypertensive rats compared to the level expressed in sham. iTRAQ-based quantitative proteomics analysis revealed that nNOS regulates phosphorylations of ∼20 proteins in cardiac myofilaments. Collectively, we provide direct evidence that different splice variants of nNOS are expressed in myofilament and membrane/cytosol fractions of cardiomyocytes. Discrete expressions of various splice variants in different compartments of cardiomyocytes suggest diverse roles nNOS play in healthy and diseased heart.

Enzyme Feed Additive with Arazyme Improve Growth Performance, Meat Quality, and Gut Microbiome of Pigs.

The supplementation of pig diets with exogenous enzymes is widely used with the expectation that it will improve the efficiency of nutrient utilization, thereby, improving growth performance. This study aims to evaluate the effects of a 0.1% (v/v) multi-enzyme (a mixture of arazyme (2,500,000 Unit/kg), xylanase (200,000 Unit/kg) and mannanase (200,000 Unit/kg)) supplementation derived from invertebrate symbiotic bacteria on pig performance. Here, 256 growing pigs were assigned to control and treatment groups, respectively. The treatment group exhibited a significantly reduced average slaughter age; the final body weight and average daily gain increased compared with that of the control group. In the treatment group, the longissimus muscle showed a remarkable decrease in cooking loss, shear force, and color values with increased essential and non-essential amino acid concentrations. Furthermore, the concentrations of mono- and polyunsaturated fatty acids in the treatment group increased. Feed additive supplementation increased the family of Ruminococcaceae and genera Lactobacillus, Limosilactobacillus, Turicibacter, and Oscillibacter, which play a positive role in the host physiology and health. Predicted metabolic pathway analysis confirmed that operational taxonomic units and predicted amino acid biosynthesis pathways were strongly associated. The results suggest that applying exogenous enzymes derived from invertebrate symbiotic bacteria enhances animal performance.

Arazyme in combination with dietary carbohydrolases influences odor emission and gut microbiome in growing-finishing pigs.

This study evaluated the effects of supplementing feed with arazyme and dietary carbohydrolases derived from invertebrate gut-associated symbionts on the noxious gas emissions, gut microbiota, and host-microbiome interactions of pigs. Here, 270 and 260 growing pigs were assigned to control and treatment groups, respectively. The tested feed additives contained a mixture of arazyme (2,500,000 Unit/kg) and synergetic enzymes, xylanase (200,000 Unit/kg) and mannanase (200,000 Unit/kg), derived from insect gut-associated symbionts in a 7.5:1:1 ratio. The control group was fed a basal diet and the treatment group was fed the basal diet supplemented with 0.1 % enzyme mixture (v/v) for 2 months. Odorous gases were monitored in ventilated air from tested houses. Fecal samples were collected from steel plate under the cage at the completion of the experiment to determine chemical composition, odor emissions, and bacterial communities. There was a significant decrease in the concentration of NH3 (22.5 vs. 11.2 ppm; P < 0.05), H2S (7.35 vs. 3.74 ppm; P < 0.05), trimethylamine (TMA) (0.066 vs. 0.001 ppm; P < 0.05), and p-cresol (0.004 ppm vs. 0 ppm; P < 0.05) at 56 d in treatment group compared with the control group. Moreover, fecal analysis results showed that exogenous enzyme supplementation caused a reduction in VFAs and indole content with approximately >60 % and 72.7 %, respectively. The result of gas emission analysis showed that NH3 (9.9 vs. 5.3 ppm; P < 0.05) and H2S (5.8 vs. 4.1 ppm; P < 0.05) were significantly reduced in the treatment group compared to the control group. The gut microbiota of the treatment group differed significantly from that of the control group, and the treatment group altered predicted metabolic pathways, including sulfur and nitrogen related metabolism, urea degradation. The results demonstrated that supplementing feed with arazyme with dietary carbohydrolases effectively controls noxious gas emissions and improves health and meat quality of pigs.

Antiviral Effects of Lindera obtusiloba Leaf Extract on Murine Norovirus-1 (MNV-1), a Human Norovirus Surrogate, and Potential Application to Model Foods.

Noroviruses are the leading cause of acute gastroenteritis and food poisoning worldwide. In this study, we investigated the anti-noroviral activity of Lindera obtusiloba leaf extract (LOLE) using murine norovirus (MNV-1), a surrogate of human norovirus. Preincubation of MNV-1 with LOLE at 4, 8, or 12 mg/mL for 1 h at 25 °C significantly reduced viral infectivity, by 51.8%, 64.1%, and 71.2%, respectively. Among LOLE single compounds, ß-pinene (49.7%), α-phellandrene (26.2%), and (+)-limonene (17.0%) demonstrated significant inhibitory effects on viral infectivity after pretreatment with MNV-1, suggesting that the anti-noroviral effects of LOLE may be due to the synergetic activity of several compounds, with ß-pinene as a key molecule. The inhibitory effect of LOLE was tested on the edible surfaces of lettuce, cabbage, and oysters, as well as on stainless steel. After one hour of incubation at 25°C, LOLE (12 mg/mL) pretreatment significantly reduced MNV-1 plaque formation on lettuce (76.4%), cabbage (60.0%), oyster (38.2%), and stainless-steel (62.8%). These results suggest that LOLE effectively inhibits norovirus on food and metal surfaces. In summary, LOLE, including ß-pinene, may inactivate norovirus and could be used as a natural agent promoting food safety and hygiene.

Additional Effect of Dietary Fiber in Patients with Type 2 Diabetes Mellitus Using Metformin and Sulfonylurea: An Open-Label, Pilot Trial.

BACKGROUND: Metformin, sulfonylurea, and dietary fiber are known to affect gut microbiota in patients with type 2 diabetes mellitus (T2DM). This open and single-arm pilot trial investigated the effects of the additional use of fiber on glycemic parameters, insulin, incretins, and microbiota in patients with T2DM who had been treated with metformin and sulfonylurea. METHODS: Participants took fiber for 4 weeks and stopped for the next 4 weeks. Glycemic parameters, insulin, incretins during mixed-meal tolerance test (MMTT), lipopolysaccharide (LPS) level, and fecal microbiota were analyzed at weeks 0, 4, and 8. The first tertile of difference in glucose area under the curve during MMTT between weeks 0 and 4 was defined as 'responders' and the third as 'nonresponders,' respectively. RESULTS: In all 10 participants, the peak incretin levels during MMTT were higher and LPS were lower at week 4 as compared with at baseline. While the insulin sensitivity of the 'responders' increased at week 4, that of the 'nonresponders' showed opposite results. However, the results were not statistically significant. In all participants, metabolically unfavorable microbiota decreased at week 4 and were restored at week 8. At baseline, metabolically hostile bacteria were more abundant in the 'nonresponders.' In 'responders,' Roseburia intestinalis increased at week 4. CONCLUSION: While dietary fiber did not induce additional changes in glycemic parameters, it showed a trend of improvement in insulin sensitivity in 'responders.' Even if patients are already receiving diabetes treatment, the additional administration of fiber can lead to additional benefits in the treatment of diabetes.

Molecular epidemiology of norovirus infections in children with acute gastroenteritis in South Korea in November 2005 through November 2006.

Norovirus infections were detected in 114 of 762 children with acute gastroenteritis in South Korea from November 2005 to November 2006. Seasonality peaks in December, March, and October were also assessed in this study. We identified seven noroviral genotypes (GI-6, GII-2, GII-3, GII-4, GII-5, GII-6, and GII-8) and a C1-120 strain showing low identity (79.3%) with GII-13 and GII-17.

Inactivation of Norovirus by Lemongrass Essential Oil Using a Norovirus Surrogate System.

This study investigated the effect of lemongrass essential oil (LGEO) on the infectivity and viral replication of norovirus. Murine norovirus 1 (MNV-1), a surrogate of human norovirus, was preincubated with LGEO and then used to infect RAW 264.7 cells in a plaque reduction assay. LGEO exhibited a significant reduction in MNV-1 plaque formation in both time- and dose-dependent manners. The quantification of viral genome by quantitative real-time PCR showed similar results in line with those of the plaque reduction assay. It was revealed that citral, a single compound in LGEO, showed dramatic reduction in MNV-1 infectivity (-73.09% when using a treatment of 0.02%, v/v). The inhibitory activity of LGEO on viral replication was further investigated in HG23 cells that harbored a human norovirus replicon. LGEO treatment significantly reduced viral replication in HG23 cells, which suggests that LGEO may have dual inhibitory activities that inactivate viral coat proteins required for viral infection and suppress norovirus genome replication in host cells. In animal experiments, oral administration of murine norovirus preincubated with LGEO significantly suppressed virus infectivity in vivo. Collectively, these results suggest that LGEO, in particular the LGEO component citral, inactivates the norovirus and its subsequent replication in host cells. Thus, LGEO shows promise as a method of inhibiting norovirus within the food industry.

Inactivation of norovirus and surrogates by natural phytochemicals and bioactive substances.

Human norovirus is the leading cause of sporadic gastroenteritis, which is responsible for more than 90% of all nonbacterial gastroenteritis outbreaks. While norovirus infections typically cause mild and self-limiting symptoms lasting 24-48 h, chronic persistent infections can cause severe symptoms. Although recent advances have been made in understanding the molecular characteristics of norovirus infection, no norovirus-specific antiviral drugs, or vaccines are available. Conventional intervention methods used to inactivate norovirus, such as treatment with disinfecting agents (e.g. ethanol, hypochlorite, and quaternary ammonium formulations), have shown a lack of efficacy against human norovirus when they are applied to foods and in food preparation processes. Therefore, alternative antiviral or inactivating agents such as phytochemicals have received attention as potential norovirus inhibitors due to their relatively low toxicity and lack of side effects, which allows them to be prepared as food-safe formulations. Evidence from studies using viral surrogates suggests that numerous phytochemicals and foods containing flavonoids and polyphenols have anti-norovirus activity, and future studies will be necessary to confirm the effectiveness of such compounds against human norovirus and the molecular mechanisms through which they produce antiviral effects.

Identification of human and animal fecal contamination after rainfall in the Han River, Korea.

We investigated the effect of rainfall on the levels and sources of microbial contamination in the Han River, Korea. Thirty-four samples were collected at two sampling sites located upstream and downstream in the river from July 2010 to February 2011. Various fecal indicator microorganisms, including total coliform, fecal coliform, Escherichia coli, Enterococcus spp., somatic and male-specific (F+) coliphage, and four major enteric viruses were analyzed. Rainfall was positively correlated with the levels of fecal coliform and norovirus at both sampling sites. Additionally, rainfall was positively correlated with the levels of total coliform, E. coli, Enterococcus spp., and F+ coliphage at the upstream site. To identify the source of fecal contamination, microbial source tracking (MST) was conducted using both male-specific (F+) RNA coliphage and the Enterococcus faecium esp gene as previously described. Our results clearly indicated that the majority of fecal contamination at the downstream Han River site was from a human source. At the upstream sampling site, contamination from human fecal matter was very limited; however, fecal contamination from non-point animal sources increased following rainfall. In conclusion, our data suggest that rainfall significantly affects the level and source of fecal contamination in the Han River, Korea.

Trends in Cervical Cancer Mortality by Socioeconomic Status in Korean Women between 1998 and 2009.

BACKGROUND: Death from uterine cervical cancer could be preventable by an active participation of women at risk in a screening program such as the Papanicolaou test. In order to examine the presence of socioeconomic disparity in preventable deaths, we evaluated the time trends of cervical cancer mortality by socioeconomic status in Korean women. METHODS: We selected level of educational attainment and marital status as surrogate indices of socioeconomic status. Using death certificate data and Korean Population and Housing Census data from Korea National Statistical office, we calculated age-standardized yearly mortality rates from cervical cancer between 1998 and 2009 according to the level of education as well as marital status. RESULTS: Cervical cancer mortality peaked in 2003 and then decreased gradually over time. Cervical cancer mortality was the highest in the group with the lowest level of educational attainment in all age groups and the gap between the lowest and the highest educational level has increased over time. Cervical cancer mortality was lower in married women than unmarried women in all age groups, and the degree of difference did not change over time. CONCLUSION: In the Korean population, socioeconomic differential in cervical cancer mortality has persisted over time.