Improved nearest-neighbor parameters for the stability of RNA/DNA hybrids under a physiological condition

The stability of Watson–Crick paired RNA/DNA hybrids is important for designing optimal oligonucleotides for ASO (Antisense Oligonucleotide) and CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)–Cas9 techniques. Previous nearest-neighbour (NN) parameters for predicting hybrid stability in a 1 M NaCl solution, however, may not be applicable for predicting stability at salt concentrations closer to physiological condition (e.g. ∼100 mM Na⁺ or K⁺ in the presence or absence of Mg²⁺). Herein, we report measured thermodynamic parameters of 38 RNA/DNA hybrids at 100 mM NaCl and derive new NN parameters to predict duplex stability. Predicted ΔG°₃₇ and T_m values based on the established NN parameters agreed well with the measured values with 2.9% and 1.1°C deviations, respectively. The new results can also be used to make precise predictions for duplexes formed in 100 mM KCl or 100 mM NaCl in the presence of 1 mM Mg²⁺, which can mimic an intracellular and extracellular salt condition, respectively. Comparisons of the predicted thermodynamic parameters with published data using ASO and CRISPR–Cas9 may allow designing shorter oligonucleotides for these techniques that will diminish the probability of non-specific binding and also improve the efficiency of target gene regulation.     

UNC-16 alters DLK-1 localization and negatively regulates actin and microtubule dynamics in Caenorhabditis elegans regenerating neurons

Neuronal regeneration after injury depends on the intrinsic growth potential of neurons. Our study shows that UNC-16, a Caenorhabditis elegans JIP3 homolog, inhibits axonal regeneration by regulating initiation and rate of regrowth. This occurs through the inhibition of the regeneration-promoting activity of the long isoform of DLK-1 and independently of the inhibitory short isoform of DLK-1. We show that UNC-16 promotes DLK-1 punctate localization in a concentration-dependent manner limiting the availability of the long isoform of DLK-1 at the cut site, minutes after injury. UNC-16 negatively regulates actin dynamics through DLK-1 and microtubule dynamics partially via DLK-1. We show that post-injury cytoskeletal dynamics in unc-16 mutants are also partially dependent on CEBP-1. The faster regeneration seen in unc-16 mutants does not lead to functional recovery. Our data suggest that the inhibitory control by UNC-16 and the short isoform of DLK-1 balances the intrinsic growth-promoting function of the long isoform of DLK-1 in vivo. We propose a model where UNC-16’s inhibitory role in regeneration occurs through both a tight temporal and spatial control of DLK-1 and cytoskeletal dynamics.     

Efficient establishment of pure cultures of yellow chanterelle Cantharellus anzutake from ectomycorrhizal root tips,and morphological characteristics of ectomycorrhizae and cultured mycelium

Species of fleshy yellow Cantharellus are known as chanterelles, which are among the most popular wild edible mycorrhizal mushrooms in the world. However, pure culture isolates of Cantharellus are rare. We report an efficient isolation technique of the Japanese golden chanterelle, Cantharellus anzutake, from its ectomycorrhizal root tips. Field-sampled fresh ectomycorrhizal root tips of C. anzutake on various hosts such as pines, spruce, and oaks were vortexed with 0.005% Tween 80 solution, surface sterilized with 1% calcium hypochlorite solution, rinsed with sterilized distilled water, and placed on modified Norkrans’ C (MNC) agar plate medium. Most ectomycorrhizal root tips of C. anzutake produced yellowish mycelial colonies within a few months. In contrast, tissue isolation from basidiomata provided limited cultures of C. anzutake but much contamination of bacteria and molds, even on media that contained antibiotics. The established C. anzutake cultures had clamp connections on the hyphae and contained intracellular oily droplets. These cultured isolates were identified as C. anzutake by sequence analysis of the rRNA internal transcribed spacer (ITS) region and translation elongation factor EF1-alpha (tef-1) genes.     

Life cycle environmental and economic impact of a food waste recycling-farming system: a case study of organic vegetable farming in Japan

Purpose

Bio-based recycling systems and agricultural production using recycled materials are often evaluated separately. This study performs an environmental and socio-economic life cycle assessment (LCA) of a food waste treatment and spinach farming system in Japan. The environmental and economic tradeoffs of introducing a recycling system and the net environmental benefit of the substitution of market fertilizer considering operation changes are also examined.

Methods

Three scenarios were developed and compared. In the conventional (CV) scenario, food waste is collected, incinerated, and disposed of in landfill, and the farmer uses market organic fertilizer. The on-site composting (OC) scenario processes food waste using an on-site garbage disposer and transports compost to a nearby spinach farmer. Food waste in the centralized composting (CC) scenario is transported to a centralized composting facility and resultant compost is sent to the farm. Primary data were obtained from field experiments and interviews. Non-greenhouse gas (GHG) emissions from the field and nitrogen leaching to water systems were simulated using the denitrification–decomposition (DNDC) model.

The environmental LCA targeted climate change, eutrophication, and waste landfill. An input–output analysis estimated socio-economic indicators, namely gross added value and employment inducement effect.

Results and discussion

The scenario with the lowest impact is the CC scenario. Climate change and eutrophication impacts are highest in the OC scenario and waste landfill impacts are most significant in the CV scenario. The weighted impact by LIME2 can be reduced by 47% in the CC scenario and 17% in the OC scenario due to the recycling of food waste instead of dumping in the landfill. The difference in socio-economic indicators between the scenarios was relatively small, although the CV scenario encouraged more employment. The substitution effect of composting, as well as the environmental impact reduction of replacing market organic fertilizer with compost, will result in 28.7% of the avoided impacts in GHG emissions.

Conclusions

Both composting scenarios are feasible from an environmental and socio-economic perspective when compared with conventional organic production, although there is a tradeoff between waste landfill and GHG emissions for the on-site composting system. However, the OC scenario needs to save electricity to improve its environmental competitiveness with the CV scenario. When considering the substitution effect of composting, it is recommended to take into account that agricultural operation also changes.

     

Type II NKT cells stimulate diet-induced obesity by mediating adipose tissue inflammation, steatohepatitis and insulin resistance

The progression of obesity is accompanied by a chronic inflammatory process that involves both innate and acquired immunity. Natural killer T (NKT) cells recognize lipid antigens and are also distributed in adipose tissue. To examine the involvement of NKT cells in the development of obesity, C57BL/6 mice (wild type; WT), and two NKT-cell-deficient strains, Jα18(-/-) mice that lack the type I subset and CD1d(-/-) mice that lack both the type I and II subsets, were fed a high fat diet (HFD). CD1d(-/-) mice gained the least body weight with the least weight in perigonadal and brown adipose tissue as well as in the liver, compared to WT or Jα18(-/-) mice fed an HFD. Histologically, CD1d(-/-) mice had significantly smaller adipocytes and developed significantly milder hepatosteatosis than WT or Jα18(-/-) mice. The number of NK1.1(+)TCRβ(+) cells in adipose tissue increased when WT mice were fed an HFD and were mostly invariant Vα14Jα18-negative. CD11b(+) macrophages (Mφ) were another major subset of cells in adipose tissue infiltrates, and they were divided into F4/80(high) and F4/80(low) cells. The F4/80(low)-Mφ subset in adipose tissue was increased in CD1d(-/-) mice, and this population likely played an anti-inflammatory role. Glucose intolerance and insulin resistance in CD1d(-/-) mice were not aggravated as in WT or Jα18(-/-) mice fed an HFD, likely due to a lower grade of inflammation and adiposity. Collectively, our findings provide evidence that type II NKT cells initiate inflammation in the liver and adipose tissue and exacerbate the course of obesity that leads to insulin resistance.     

Fatty acyl-CoA binding activity of the nuclear thyroid hormone receptor

Long-chain fatty acids and their acyl-CoA esters are potent inhibitors of nuclear thyroid hormone (T₃) receptor in vitro. In the present study, we obtained evidence for acyl-CoA binding activity in the nuclear extract from rat liver. The activity sedimented at a position (3.5 S) identical with that of the T₃ receptor, and the two activities sedimented together. Similarly, they coeluted on DEAE-Sephadex. After partial purification of the receptor, it was again inhibited strongly by acyl-CoAs. Heat stability and a partial trypsin digestion of the receptor both suggested that the action site of oleoyl-CoA overlapped the T₃-binding domain of the receptor. In addition, thyroid hormone receptor β1, synthesized in vitro, bound oleoyl-CoA specifically and its T₃-binding activity was inhibited. The dissociation constant for oleoyl-CoA binding to the partially purified receptor was 1.2 × 10^?7 M. This value as well as its molecular size distinguished the nuclear binding sites from the cytoplasmic fatty acid/acyl-CoA binding proteins. Oleoyl-CoA had no effect on the glucocorticoid receptor, another member of the nuclear hormone-receptor superfamily. From these results, we propose that thyroid hormone receptor is a specific acyl-CoA binding protein of the cell nucleus.     

Aggregatibacter actinomycetemcomitans induces detachment and death of human gingival epithelial cells and fibroblasts via elastase release following leukotoxin‐dependent neutrophil lysis

Aggregatibacter actinomycetemcomitans is considered to be associated with periodontitis. Leukotoxin (LtxA), which destroys leukocytes in humans, is one of this bacterium's major virulence factors. Amounts of neutrophil elastase (NE), which is normally localized in the cytoplasm of neutrophils, are reportedly increased in the saliva of patients with periodontitis. However, the mechanism by which NE is released from human neutrophils and the role of NE in periodontitis is unclear. In the present study, it was hypothesized that LtxA induces NE release from human neutrophils, which subsequently causes the breakdown of periodontal tissues. LtxA‐treatment did not induce significant cytotoxicity against human gingival epithelial cells (HGECs) or human gingival fibroblasts (HGFs). However, it did induce significant cytotoxicity against human neutrophils, leading to NE release. Furthermore, NE and the supernatant from LtxA‐treated human neutrophils induced detachment and death of HGECs and HGFs, these effects being inhibited by administration of an NE inhibitor, sivelestat. The present results suggest that LtxA mediates human neutrophil lysis and induces the subsequent release of NE, which eventually results in detachment and death of HGECs and HGFs. Thus, LtxA‐induced release of NE could cause breakdown of periodontal tissue and thereby exacerbate periodontitis.     

Evaluation of the allergenicity of ω5-gliadin-deficient Hokushin wheat (1BS-18) in a wheat allergy rat model

We previously developed Hokushin wheat line as a hypoallergenic wheat lacking ω5-gliadin (1BS-18), a major allergen for wheat-dependent exercise-induced anaphylaxis. However, the allergenicity of 1BS-18 has not been understood completely. In this study, we evaluated the allergenicity of 1BS-18 such as anaphylactic elicitation ability and sensitization ability using rats sensitized with ω5-gliadin or glutens prepared from Hokushin (Hokushin gluten) or 1BS-18 (1BS-18 gluten). Rats were sensitized by intraperitoneal administration of ω5-gliadin, Hokushin gluten or 1BS-18 gluten. Immunoglobulin E-mediated systemic anaphylaxis was evaluated by measuring changes in rectal temperature for 30 min after intravenous challenge with ω5-gliadin or the test glutens in unsensitized rats or rats sensitized with ω5-gliadin or the test glutens. In ω5-gliadin-sensitized rats, intravenous challenge with ω5-gliadin or Hokushin gluten significantly decreased the rectal temperature at 30 min after challenge while challenge with 1BS-18 gluten did not reduce the rectal temperature. Furthermore, intravenous challenge with ω5-gliadin significantly decreased the rectal temperature in rats sensitized with Hokushin gluten or 1BS-18 gluten. However, the reduced degree observed in 1BS-18 gluten-sensitized rats was smaller than that in Hokushin gluten-sensitized rats. In conclusion, 1BS-18 elicited no allergic reaction in ω5-gliadin-sensitized rats and had less sensitization ability for ω5-gliadin than that of Hokushin wheat.     

Effects of losartan on the collagen degradative enzymes in hypertrophic and congestive types of cardiomyopathic hamsters

The present study was undertaken to determine the effects of AT1 receptor blockade which occurred in response to losartan, on the extracellular matrix (ECM) degradation process in the Bio 14.6 (n = 12) and Bio 53.58 (n = 12) strains which are referred as models of hypertrophic and dilated cardiomyopathy, respectively. The administration of losartan (30 mg/kg/day) in hamsters from 10–20 weeks of age reduced the accumulation of the left ventricular collagen matrix in both of the Bio 14.6 and the Bio 53.58 strains. According to the RTPCR, the levels of mRNA for matrix metalloproteinase (MMP) and the tissue inhibitor of MMP (TIMP) were examined. MMP1, 2, 3, and 9 were more enhanced in both myopathic strains than in the control F1 strains. With losartan, the levels of MMP1, 2, 9, TIMP1 and 2 decreased in the both strains but those for MMP3 did not in Bio 14.6 strains. TIMP3 and 4 mRNA levels did not change in any of the experimental hamsters, whether treated or untreated with losartan. The Western blots also showed similar observations in the both strains as seen in mRNA expressions although MMP2 in the Bio 53.58 strains did not differ between treated and untreated with losartan. Although losartan has an inhibitory effect on collagen accumulation in the development of cardiomyopathy, MMPs (1, 2, 9) and TIMPs (1, 2) seem to be susceptible to responding to losartan in Bio cardiomyopathic hamsters.     

Rapid shifting of foraging pattern by Yakushima macaques ( Macaca fuscata yakui ) in response to heavy fruiting of Myrica rubra

We describe short-term changes in foraging behavior by wild Yakushima macaques (Macaca fuscata yakui),which inhabit a warm-temperate broad—leaved forest on Yakushima Island (30°N, 131°E), Japan. Rapid changes of dietary composition, activity budget, and range use by the monkeys occurred from May to June, apparently associated with changes in the availability of the fruit of Myrica rubraBefore the fruit ripened, monkeys spent less time moving and more time feeding on many species of leaves, which accounted for 40% of feeding time. However, when M. rubrabegan to ripen, they fed intensively on the fruit, which accounted for three-fourths of feeding time,though the activity budget remained unaffected As fiuit of M. rubradecreased,the monkeys fed more on the fruit of other species and on insects, and spent more time moving at higher speeds. There marked shifts in foraging pattern occurred within only two months. In terms of moving cost and dietary quality,Yakushima macaques shifted their foraging pattern according to the availability of M. rubrafrom a “low-cost, low-yield” strategy to a “low-cost, high-yield” strategy, and then to a more costly strategy. The ability to make such rapid shifts in foraging pattern may allow the macaques to effectively use the highly variable food supply within their small range.