An inhibitor persistently decreased enteric methane emission from dairy cows with no negative effect on milk production

A quarter of all anthropogenic methane emissions in the United States are from enteric fermentation, primarily from ruminant livestock. This study was undertaken to test the effect of a methane inhibitor, 3-nitrooxypropanol (3NOP), on enteric methane emission in lactating Holstein cows. An experiment was conducted using 48 cows in a randomized block design with a 2-wk covariate period and a 12-wk data collection period. Feed intake, milk production, and fiber digestibility were not affected by the inhibitor. Milk protein and lactose yields were increased by 3NOP. Rumen methane emission was linearly decreased by 3NOP, averaging about 30% lower than the control. Methane emission per unit of feed dry matter intake or per unit of energy-corrected milk were also about 30% less for the 3NOP-treated cows. On average, the body weight gain of 3NOP-treated cows was 80% greater than control cows during the 12-wk experiment. The experiment demonstrated that the methane inhibitor 3NOP, applied at 40 to 80 mg/kg feed dry matter, decreased methane emissions from high-producing dairy cows by 30% and increased body weight gain without negatively affecting feed intake or milk production and composition. The inhibitory effect persisted over 12 wk of treatment, thus offering an effective methane mitigation practice for the livestock industries.The livestock sector is a significant source of greenhouse gas (GHG) emissions in the United States and globally (1, 2). In the United States, enteric fermentation of feed by ruminants is the largest source of anthropogenic methane emissions (0.14 Gt of CO₂ Eq. in 2012; or 25% of the total methane emissions; ref. 3). Globally, according to the most recent Intergovernmental Panel on Climate Change (IPCC) report, GHG emissions from agriculture represent around 10–12% (5.0–5.8 Gt CO₂ Eq/yr) of the total anthropogenic GHG emissions (1). In this report, livestock contribution to the global anthropogenic GHG emissions was estimated at 6.3%, with GHG emissions from enteric fermentation accounting for 2.1 Gt CO₂ Eq/yr and manure management accounting for 0.99 Gt CO₂ Eq/yr (1). The relative contribution of emissions from enteric fermentation to the total agricultural GHG emissions will vary by region depending on the structure of agricultural production and type of livestock production systems. For example, GHG from enteric fermentation were estimated at 57% for New Zealand, a country with a large, pasture-based livestock sector (4). Extensive research in recent years has provided a number of viable enteric methane mitigation practices, such as alternative electron receptors, methane inhibitors, dietary lipids, and increased animal productive efficiency (5). Methane emission in the reticulo-rumen is an evolutionary adaptation that enables the rumen ecosystem to dispose of hydrogen, a fermentation product and an important energy substrate for the methanogenic archaea (6), which may otherwise accumulate and inhibit carbohydrate fermentation and fiber degradation (7, 8). Some compounds may be effective in decreasing methane emission, but they may also decrease feed intake, fiber degradability, and animal productivity (5), or the rumen archaea may adapt to them (9). Therefore, it is important to evaluate methane mitigation strategies in long-term experiments, which for livestock experimentation requires treatment periods considerably longer than the 21–28 d, common for crossover designs. In addition, due to a variety of constraints and confounding factors of batch or continuous culture in vitro systems (5, 10), mitigation compounds, including methane inhibitors, have to be tested in vivo using animals with similar productivity to those on commercial farms. An example of the limitations of in vitro systems is a series of experiments with garlic oil. In continuous rumen culture, garlic oil was very effective in inhibiting rumen methane emission (11), but it failed to produce an effect in sheep (12). The nutrient requirements of high-producing dairy cows are much greater than those of nonlactating or low-producing cows (13) and hence any reduction in feed intake caused by a methane mitigation compound or practice would likely result in decreased productivity, which may not be evident in low-producing cows.Methane inhibitors are chemical compounds with inhibitory effects on rumen archaea. Compounds such as bromochloromethane, 2-bromoethane sulfonate, chloroform, and cyclodextrin have been tested, some successfully, in various ruminant species (5). Inhibition of methanogenesis by these compounds in vivo can be up to 60% with the effect of bromochloromethane shown to persist in long-term experiments (5, 14). However, the viability of these compounds as mitigation agents has been questioned due to concerns for animal health, food safety, or environmental impact. Bromochloromethane, for example, is an ozone-depleting agent and is banned in many countries.Among the efficacious methane inhibitors identified is 3-nitrooxypropanol (3NOP; ref. 15). This compound was part of a developmental program designing specific small molecule inhibitors for methyl coenzyme-M (CoM) reductase, the enzyme that catalyzes the last step of methanogenesis, the reduction of methyl CoM and coenzyme-B (CoB) into methane and a CoM–CoB complex (16). A continuous in vitro culture study (11) was followed by in vivo experiments in sheep (17), beef (18), and dairy cattle (19, 20), which demonstrated that 3NOP is an effective methane inhibitor. However, these experiments were conducted using nonlactating animals (17), or were short-term (<35 d; refs. 19 and 20). The rumen microorganisms have the ability to adapt to foreign agents or changes in the feeding regimen and, therefore, short-term responses are not representative of the effect of a given mitigation compound or practice in real farm conditions. McIntosh et al. (21), for example, showed that the MIC50 of essential oils doubled or tripled for a number of important rumen bacteria (Butyrivibrio fibrisolvens, Prevotella bryantii, Ruminococcus albus, Ruminobacter amylophilus), if they were adapted to the treatment for a period of 10 d. Thus, it is critically important for the success of GHG mitigation efforts to substantiate the mitigation potential of a given compound in long-term animal experiments before considering it for adoption by the livestock industries.     

BRAF mutation testing with a rapid,fully integrated molecular diagnostics system

Fast and accurate diagnostic systems are needed for further implementation of precision therapy of BRAF-mutant and other cancers. The novel Idylla^TM BRAF Mutation Test has high sensitivity and shorter turnaround times compared to other methods. We used Idylla to detect BRAF V600 mutations in archived formalin-fixed paraffin-embedded (FFPE) tumor samples and compared these results with those obtained using the cobas 4800 BRAF V600 Mutation Test or MiSeq deep sequencing system and with those obtained by a Clinical Laboratory Improvement Amendments (CLIA)-certified laboratory employing polymerase chain reaction–based sequencing, mass spectrometric detection, or next-generation sequencing. In one set of 60 FFPE tumor samples (15 with BRAF mutations per Idylla), the Idylla and cobas results had an agreement of 97%. Idylla detected BRAF V600 mutations in two additional samples. The Idylla and MiSeq results had 100% concordance. In a separate set of 100 FFPE tumor samples (64 with BRAF mutation per Idylla), the Idylla and CLIA-certified laboratory results demonstrated an agreement of 96% even though the tests were not performed simultaneously and different FFPE blocks had to be used for 9 cases. The Idylla^TM BRAF Mutation Test produced results quickly (sample to results time was about 90 minutes with about 2 minutes of hands on time) and the closed nature of the cartridge eliminates the risk of PCR contamination. In conclusion, our observations demonstrate that the Idylla test is rapid and has high concordance with other routinely used but more complex BRAF mutation–detecting tests.     

Overexpression of H- and L-ferritin subunits in lens epithelial cells: Fe metabolism and cellular response to UVB irradiation.

PURPOSE. To determine the effect of changes in ferritin subunit makeup on Fe metabolism and the resistance of lens epithelial cells (LEC) to photo-oxidative stress. METHODS. Cultured canine LEC were transiently transfected with pTargeT mammalian expression vector containing the whole coding sequence of H- or L-chain cDNA. The subunit composition of newly synthesized ferritin was analyzed by metabolic labeling and SDS-PAGE electrophoresis. Total ferritin concentration was measured by ELISA. Fe uptake and incorporation into ferritin was determined by incubating transfected cells with (59)Fe-labeled transferrin followed by native PAGE electrophoresis. The effect of UV irradiation was assessed by cell count after exposure of transfected cells to UVB radiation. RESULTS. Transfected cells differentially expressed H- and L-ferritin chains from cDNA under the control of CMV promoter; overexpression of L-chain was much greater than that of H-chain. The expressed chains assembled into ferritin molecules under in vitro and in vivo condition. The ferritin of H-transfectants incorporated significantly more Fe than those of L-transfectants. The UVB irradiation reduced cell number of L-transfectants by half, whereas H-chain transfectants were protected. CONCLUSIONS. Post-transfectional expression of ferritin H- and L-chains in LEC appears to be regulated differentially. Overexpression of L-chain ferritin did not have a major effect on cellular Fe distribution and did not protect LEC against UV irradiation, whereas overexpression of H-chain resulted in increased storage of Fe in ferritin and protected cells from UV damage.     

Perceptive properties of the multi-site electrical microstimulation of the olfactory bulb in the rat

Electrical microstimulation of the olfactory bulb in different locations has been shown to provide water-deprived rats with discriminative cues for selecting a palatable solution without tasting it in a two-choice test. Some perceptive properties of bulbar electrical stimulation were investigated. It was shown that the perceptive effect evoked by stimulating a given site could be recognized when this site was stimulated together with several others. The animals' perception of multi-site stimulation patterns seems therefore to be analytical rather than synthetic. Discrimination of stimulation patterns did not require presentation of concurrent patterns inside a short time interval. Identification of a multi-site pattern was possible when this pattern was presented alone in a test session. Individual characteristics of bulbar microstimulation appear to be perceived absolutely rather than differentially. A good retention of the discrimination learning of specific stimulation patterns was observed. Animals could identify stimulation patterns after complete interruption of the training for 17 days. The results are discussed with reference to the properties of the natural stimulation of the olfactory system.     

Increased sensitivity to etomidate in the elderly: initial distribution versus altered brain response

To determine the effect of aging on the pharmacokinetics and pharmacodynamics of etomidate, we administered etomidate (5 to 10 mg/min) by intravenous infusion to 21 healthy surgical patients, age 22 to 82 yr. Etomidate produced progressive slowing of the EEG to an easily recognized pattern (stage 3) that determined the dosage endpoint. Subsequent power-spectrum analysis of the EEG gave the median frequency. Median frequency values and simultaneous measurements of blood etomidate concentration were incorporated into a sigmoid Emax pharmacodynamic model that permitted an estimate of IC50, the blood etomidate concentration which produced a 50% reduction in the median frequency. The dose of etomidate required to reach the uniform EEG endpoint decreased significantly with increasing age (r2 = .68) as did the dose needed to produce maximal median frequency depression (r2 = .69). None of the parameters of the pharmacodynamic effect model, including IC50, correlated with age, suggesting that increased brain sensitivity in the elderly does not cause the age-related change in dose requirement. The initial distribution volume for etomidate decreased significantly with increasing age (r = .56), implying that a higher initial blood concentration in the elderly following any given dose of etomidate is part of the cause of the lower dose requirement in the elderly patient. A contracted initial distribution volume in the elderly may result from well described physiologic changes of age. Etomidate clearance also decreased with age. Age-dependent changes in etomidate pharmacokinetics rather than altered brain responsiveness may be the basis for the decreased etomidate dose requirement in the elderly.     

The utility of a structured evaluation of elderly patients for continuous peritoneal dialysis.

The elderly comprise an increasing proportion of chronic dialysis patients. Recruiting them for continuous peritoneal dialysis (CPD) would help CPD programs maintain a patient population. We retrospectively studied the ability of a prospective evaluation to predict success with CPD in elderly (age greater than 60 years) patients. PD nurses and a renal social worker assigned scores in 10 categories, which were then averaged to obtain an over-all evaluation score. Scores were from 1-5 with 1 = good, 5 = poor, and 3 = average. Thirty-four elderly patients began CPD during the study period. Evaluation scores were available for 28 of these patients before they began dialysis. Evaluation scores less than 3 predicted success with CPD (2.2 +/- 0.2 versus 3.2 +/- 0.4 in patients transferring to hemodialysis, p less than 0.02). Patient motivation and preference were the categories that predicted success with CPD. Elderly patients were more likely than younger patients (those less than 60 years of age) to decline CPD for social reasons (46% versus 4% respectively, p less than 0.001). Elderly patients required more CPD training time than young patients (4.9 +/- 0.7 days versus 3.3 +/- 0.8 days respectively, p less than 0.01). We conclude that a prospective assessment of elderly patients can predict success with CPD and provide information important to individual structuring of CPD training and follow-up.