Search Results (28)

Structural insights into methyl-coenzyme M reductase from Methanobrevibacter ruminantium (M. ruminantium) has implications for methane mitigation strategies. Methanogenesis in ruminants is a major contributor to global greenhouse gas emissions, primarily driven by the rumen archaeon M. ruminantium. Central to this process is methyl-coenzyme M reductase (Mcr), an enzyme that catalyzes the final step of methane production. Despite its significance as a chemogenetic target for methane mitigation, the high-resolution structure of M. ruminantium Mcr has remained elusive. Here, we employed homology modeling and CDOCKER simulations within the CHARMM force field to elucidate the structural and functional features of the M. ruminantium Mcr/ligand complexes. We characterized two distinct states: the reduced Mcr_oxi-silent state bound to HS-CoM and CoB-SH, and the oxidized Mcr_silent state bound to the heterodisulfide CoM-S-S-CoB. Alanine-scanning mutagenesis identified 71 and 62 key residues per active site for each state, respectively, revealing the fundamental determinants of structural stability and substrate selectivity on the Ni-F₄₃₀ cofactor. Furthermore, structure-based pharmacophore modeling defined essential features (AAADDNNN and AAADDNN) that drive ligand binding. These findings provide a high-resolution molecular framework for the rational design of specific Mcr inhibitors, offering a robust starting point for developing broad-spectrum strategies to suppress enteric methane emissions. Full article

The effects of fallow season water and straw management on methane (CH₄) emissions during the fallow season and the subsequent rice-growing season are rarely reported, and the underlying microbial mechanisms remain unclear. A field experiment was conducted with four treatments: (1) fields flooded in both the fallow and rice seasons (FF), (2) fields drained in the fallow season and flooded in the rice season (DF), (3) FF with straw retention (FFS), and (4) DF with straw retention (DFS). The CH₄ emissions in fields under different water and straw treatments were monitored using the static closed chamber method. Methanogenic and methanotrophic communities in these fields were examined using terminal restriction fragment length polymorphism (T-RFLP) analysis based on the mcrA gene and pmoA gene encoding methyl coenzyme M reductase and particulate methane monooxygenase, respectively. The results showed that CH₄ emissions were significantly affected by water management, straw retention, season, and their interactions. Over 80% of CH₄ emissions occurred during the rice season. Field drainage during the fallow season reduced CH₄ emissions by 47.0% and 53.8% with and without straw during the rice season, respectively. Water management altered the abundance and composition of methanogens and methanotrophs, whereas the effects of straw retention were less pronounced. The quantitative polymerase chain reaction (qPCR) assay revealed that field drainage in the fallow season decreased the mcrA gene abundance by 30.0% and 23.2% with and without straw in rice season, respectively, and increased the pmoA gene abundance by 108.9% and 213.7% with and without straw in rice season, respectively. CH₄ flux was significantly positively associated with mcrA gene copy number and the ratio of mcrA to pmoA gene copy number, whereas it was significantly negatively correlated with the pmoA gene copy number. Results indicated that fallow drainage greatly decreased CH₄ emission not only during the fallow season but also during the subsequent rice season by altering the community composition of methanogens and methanotrophs. These findings provide scientific insight into the role of water and straw management in controlling CH₄ emissions through microbial community dynamics. Full article

3-Nitrooxypropanol (3-NOP) is a nitrooxy compound that specifically targets methyl-coenzyme M reductase (MCR), ultimately resulting in a reduction in methane production. In this study, we undertook an in vitro investigation of the effects of different dosages of 3-NOP on ruminal fermentation parameters, methane production, and the microbial community. A single-factor completely randomized design was adopted, comprising a control treatment (C), where no 3-NOP was added to the fermentation substrate, and three 3-NOP treatments, where 0.025 mg (low-dose treatment, LD), 0.05 mg (medium-dose treatment, MD), or 0.1 mg (high-dose treatment, HD) was added to 1 g of fermentation substrate (DM basis), followed by incubation for 24 h in vitro. The results showed that, compared with the control treatment, the three dosages of 3-NOP reduced total gas production, methane production, and acetate production (all p < 0.01). In contrast, 3-NOP treatment increased H₂ production and the molar proportions of propionate and butyrate (all p ≤ 0.02), resulting in a decrease in the acetate-propionate ratio (p < 0.01). Meanwhile, the microbial profiles were not altered by the treatments, but the relative abundances of Prevotella, Methanobrevibacter, and Ophryoscolex were increased by the MD and HD treatments (all p < 0.01), whereas those of Methanosarcina, Methanosaeta, Sphaerochaeta, RFN20, Entodinium, and Diplodinium were decreased by the HD treatment (all p ≤ 0.03). Moreover, the results of a correlation analysis showed that there was a certain correlation between these microorganisms and total gas production, methane production, H₂ production, acetate, propionate, and butyrate. In summary, under in vitro conditions, the addition of 3-NOP to the diet affected the microbial community structure, thereby altering the ruminal fermentation pattern and reducing methane production. Our results indicated that 0.05 mg per g of dietary DM is the recommended inclusion ratio for 3-NOP in the diet of lambs. Full article

Metastatic melanoma has a very poor prognosis. Statins, 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase (HMGCR) inhibitors, are cholesterol-lowering agents with a potential for cancer treatment. The inhibition of HMGCR by statins, however, induces feedback, which paradoxically upregulates HMGCR expression via sterol regulatory element-binding protein-2 (SREBP2). Dipyridamole, an antiplatelet agent, is known to inhibit SREBP2 upregulation. We aimed to demonstrate the efficacy of statin–dipyridamole combination treatment in both human and spontaneously occurring canine melanoma cell lines. The half maximal inhibitory concentration (IC₅₀) of atorvastatin showed a 68–92% reduction when combined with dipyridamole, compared with that of atorvastatin alone. In some melanoma cell lines, cell proliferation was suppressed to almost zero by the combination treatment (≥3 μM atorvastatin). Finally, the BRAF inhibitor, vemurafenib, further potentiated the effects of the combined statin–dipyridamole treatment in BRAF V600E mutation-bearing human melanoma cell lines. In conclusion, the inexpensive and frequently prescribed statin–dipyridamole combination therapy may lead to new developments in the treatment of melanoma and may potentiate the effects of vemurafenib for the targeted therapy of BRAF V600E-mutation bearing melanoma patients. The concordance between the data from canine and human melanoma cell lines reinforces this possibility. Full article

Crop straw and N fertilizer applications impact paddy rice yield and greenhouse gas (GHG) emissions. However, their interactive effects have not been well documented. This study investigated the effects of straw (S), no straw incorporation (NS), and three levels of N fertilization rates (N0, N1, and N2) on single rice (SR), double rice (DR), and rice-wheat (RW) cropping systems. Straw incorporation significantly increased total CH₄ emissions by 118.6%, 8.0%, and 79.0% in the SR, DR, and RW, respectively, compared to the NS. The total GHG emissions in DR are significantly 72.6% and 83.5% higher than those in RW and SR, respectively. Compared to NS, straw incorporation significantly increased yield-scaled emissions by 27.8%, 15.0%, and 89.0% in SR, DR, and RW, respectively. Straw with N application significantly increased average rice yield over N1 and N2 by 39.4%, 50.0%, and 6.7% in SR, DR, and RW, respectively. There was a significant correlation between methyl coenzyme M reductase (mcrA) and CH₄ emissions in r_SR = 0.87 (p < 0.05) and r_RW = 0.85 (p < 0.05), except in r_DR = 0.06 (p > 0.05). This study scientifically supports straw incorporation combined with a moderate N application rate in rice-based cropping systems to maintain high rice yields and mitigate GHG emissions. Full article

In our search for bioactive components, various chromatographic separations of the organic fractions from Filipendula glaberrima leaves led to the isolation of a new ellagitannin and a triterpenoid, along with 26 known compounds. The structures of the isolates were determined based on their spectroscopic properties and chemical evidence, which were then evaluated for their antioxidant activities, inhibitory activities on 3-hydroxy-3-methylglutaryl-coenzyme A reductase, and foam cell formation in THP-1 cells to prevent atherosclerosis. Rugosin B methyl ester (1) showed the best HMG-CoA reductase inhibition and significantly reduced ox-low-density lipoprotein-induced THP-1 macrophage-derived foam cell formation at 25 µM. In addition, no cytotoxicity was observed in THP-1 cells at 50 μg/mL of all extracts in the macrophage foam cell formation assay. Therefore, F. glaberrima extract containing 1 is promising in the development of dietary supplements due to its potential behavior as a novel source of nutrients for preventing and treating atherosclerosis. Full article

The Guaymas Basin in the Gulf of California is characterized by active seafloor spreading, the rapid deposition of organic-rich sediments, steep geothermal gradients, and abundant methane of mixed thermogenic and microbial origin. Subsurface sediment samples from eight drilling sites with distinct geochemical and thermal profiles were selected for DNA extraction and PCR amplification to explore the diversity of methane-cycling archaea in the Guaymas Basin subsurface. We performed PCR amplifications with general (mcrIRD), and ANME-1 specific primers that target the alpha (α) subunit of methyl coenzyme M reductase (mcrA). Diverse ANME-1 lineages associated with anaerobic methane oxidation were detected in seven out of the eight drilling sites, preferentially around the methane-sulfate interface, and in several cases, showed preferences for specific sampling sites. Phylogenetically, most ANME-1 sequences from the Guaymas Basin subsurface were related to marine mud volcanoes, seep sites, and the shallow marine subsurface. The most frequently recovered methanogenic phylotypes were closely affiliated with the hyperthermophilic Methanocaldococcaceae, and found at the hydrothermally influenced Ringvent site. The coolest drilling site, in the northern axial trough of Guaymas Basin, yielded the greatest diversity in methanogen lineages. Our survey indicates the potential for extensive microbial methane cycling within subsurface sediments of Guaymas Basin. Full article

Our investigation intended to analyze the effects of sulfated polysaccharides from Caulerpa racemosa (SPCr) in attenuating obesity-induced cardiometabolic syndrome via regulating the protein arginine N-methyltransferase 1-asymmetric dimethylarginine-dimethylarginine dimethylamino-hydrolase (PRMT1-DDAH-ADMA) with the mammalian target of rapamycin-Sirtuin 1–5′ AMP-activated protein kinase (mTOR-SIRT1-AMPK) pathways and gut microbiota modulation. This is a follow-up study that used SPs from previous in vitro studies, consisting of 2,3-di-O-methyl-1,4,5-tri-O-acetylarabinitol, 2,3,4,6-tetra-O-methyl-D-mannopyranose, and type B ulvanobiuronicacid 3-sulfate. A total of forty rats were randomly divided into four treatment groups: Group A received a standard diet; Group B was provided with a diet enriched in cholesterol and fat (CFED); and Groups C and D were given the CFED along with ad libitum water, and daily oral supplementation of 65 or 130 mg/kg of body weight (BW) of SPCr, respectively. Group D showed the lowest low-density lipoprotein, triglyceride, total cholesterol, and blood glucose levels, and the highest HDL level compared to the other groups in this study. These results in the group fed high-dose SPCr demonstrated a significant effect compared to the group fed low-dose SPCr (p < 0.0001), as well as in total cholesterol and blood glucose (p < 0.05). Supplementation with SPCr was also observed to have an upregulation effect on peroxisome proliferator-activated receptor gamma coactivator (PGC)-1alpha, interleukin 10, Sirtuin 1, DDAH-II, superoxide dismutase (SOD) cardio, and AMPK, which was also followed by a downregulation of PRMT-1, TNF-α, 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor, and mTOR. Interestingly, gut microbiota modulation was also observed; feeding the rats with a cholesterol-enriched diet shifted the gut microbiota composition toward the Firmicutes level, lowered the Bacteroidetes level, and increased the Firmicutes level. A dose of 130 mg/kg BW of SPCr is the recommended dose, and investigation still needs to be continued in clinical trials with humans to see its efficacy at an advanced level. Full article

Knowledge regarding the diversity of methanogenic archaeal communities in hypersaline environments is limited because of the lack of efficient cultivation efforts as well as their low abundance and metabolic activities. In this study, we explored the microbial communities in hypersaline microbial mats. Bioinformatic analyses showed significant differences among the archaeal community structures for each studied site. Taxonomic assignment based on 16S rRNA and methyl coenzyme-M reductase (mcrA) gene sequences, as well as metagenomic analysis, corroborated the presence of Methanosarcinales. Furthermore, this study also provided evidence for the presence of Methanobacteriales, Methanomicrobiales, Methanomassiliicoccales, Candidatus Methanofastidiosales, Methanocellales, Methanococcales and Methanopyrales, although some of these were found in extremely low relative abundances. Several mcrA environmental sequences were significantly different from those previously reported and did not match with any known methanogenic archaea, suggesting the presence of specific environmental clusters of methanogenic archaea in Guerrero Negro. Based on functional inference and the detection of specific genes in the metagenome, we hypothesised that all four methanogenic pathways were able to occur in these environments. This study allowed the detection of extremely low-abundance methanogenic archaea, which were highly diverse and with unknown physiology, evidencing the presence of all methanogenic metabolic pathways rather than the sheer existence of exclusively methylotrophic methanogenic archaea in hypersaline environments. Full article

Pyranoanthocyanins have been reported to possess better chemical stability and bioactivities than monomeric anthocyanins in some aspects. The hypocholesterolemic activity of pyranoanthocyanins is unclear. In view of this, this study was conducted to compare the cholesterol-lowering activities of Vitisin A with the anthocyanin counterpart Cyanidin-3-O-glucoside(C3G) in HepG2 cells and to investigate the interaction of Vitisin A with the expression of genes and proteins associated with cholesterol metabolism. HepG2 cells were incubated with 40 μM cholesterol and 4 μM 25-hydroxycholeterol with various concentrations of Vitisin A or C3G for 24 h. It was found that Vitisin A decreased the cholesterol levels at the concentrations of 100 μM and 200 μM with a dose–response relationship, while C3G exhibited no significant effect on cellular cholesterol. Furthermore, Vitisin A could down-regulate 3-hydroxy-3-methyl-glutaryl coenzyme A reductase (HMGCR) to inhibit cholesterol biosynthesis through a sterol regulatory element-binding protein 2 (SREBP2)-dependent mechanism, and up-regulate low-density lipoprotein receptor (LDLR) and blunt the secretion of proprotein convertase subtilisin/kexin type 9 (PCSK9) protein to promote intracellular LDL uptake without LDLR degradation. In conclusion, Vitisin A demonstrated hypocholesterolemic activity, by inhibiting cholesterol biosynthesis and enhancing LDL uptake in HepG2 cells. Full article

Methanogenic archaea are important organisms in the global carbon cycle that grow by producing methane gas. Methanosarcina acetivorans is a methanogenic archaeum that can grow using methylated compounds, carbon monoxide, or acetate and produces renewable methane as a byproduct. However, there is limited knowledge of how combinations of substrates may affect metabolic fluxes in methanogens. Previous studies have shown that heterodisulfide reductase, the terminal oxidase in the electron transport system, is an essential enzyme in all methanogens. Deletion of genes encoding the nonessential methylotrophic heterodisulfide reductase enzyme (HdrABC) results in slower growth rate but increased metabolic efficiency. We hypothesized that increased sulfide, supplementation of mercaptoethanesulfonate (coenzyme M, CoM-SH), or acetate would metabolically alleviate the effect of the ΔhdrABC mutation. Increased sulfide improved growth of the mutant as expected; however, supplementation of both CoM-SH and acetate together were necessary to reduce the effect of the ΔhdrABC mutation. Supplementation of CoM-SH or acetate alone did not improve growth. These results support our model for the role of HdrABC in methanogenesis and suggest M.acetivorans is more efficient at conserving energy when supplemented with acetate. Our study suggests decreased Hdr enzyme activity can be overcome by nutritional supplementation with sulfide or coenzyme M and acetate, which are abundant in anaerobic environments. Full article

Analyses of protein structures have shown the existence of molecular channels in enzymes from Prokaryotes. Those molecular channels suggest a critical role of spatial voids in proteins, above all, in those enzymes functioning under high temperature. It is expected that these spaces within the protein structure are required to access the active site and to maximize availability and thermal stability of their substrates and cofactors. Interestingly, numerous substrates and cofactors have been reported to be highly temperature-sensitive biomolecules. Methanogens represent a singular phylogenetic group of Archaea that performs anaerobic respiration producing methane during growth. Methanogens inhabit a variety of environments including the full range of temperatures for the known living forms. Herein, we carry out a dimensional analysis of molecular tunnels in key enzymes of the methanogenic pathway from methanogenic Archaea growing optimally over a broad temperature range. We aim to determine whether the dimensions of the molecular tunnels are critical for those enzymes from thermophiles. Results showed that at increasing growth temperature the dimensions of molecular tunnels in the enzymes methyl-coenzyme M reductase and heterodisulfide reductase become increasingly restrictive and present strict limits at the highest growth temperatures, i.e., for hyperthermophilic methanogens. However, growth at lower temperature allows a wide dimensional range for the molecular spaces in these enzymes. This is in agreement with previous suggestions on a potential major role of molecular tunnels to maintain biomolecule stability and activity of some enzymes in microorganisms growing at high temperatures. These results contribute to better understand archaeal growth at high temperatures. Furthermore, an optimization of the dimensions of molecular tunnels would represent an important adaptation required to maintain the activity of key enzymes of the methanogenic pathway for those methanogens growing optimally at high temperatures. Full article

In pediatric rhabdomyosarcoma (RMS), elevated Akt signaling is associated with increased malignancy. Here, we report that expression of a constitutively active, myristoylated form of Akt1 (myrAkt1) in human RMS RD cells led to hyperactivation of the mammalian target of rapamycin (mTOR)/70-kDa ribosomal protein S6 kinase (p70S6K) pathway, resulting in the loss of both MyoD and myogenic capacity, and an increase of Ki67 expression due to high cell mitosis. MyrAkt1 signaling increased migratory and invasive cell traits, as detected by wound healing, zymography, and xenograft zebrafish assays, and promoted repair of DNA damage after radiotherapy and doxorubicin treatments, as revealed by nuclear detection of phosphorylated H2A histone family member X (γH2AX) through activation of DNA-dependent protein kinase (DNA-PK). Treatment with synthetic inhibitors of phosphatidylinositol-3-kinase (PI3K) and Akt was sufficient to completely revert the aggressive cell phenotype, while the mTOR inhibitor rapamycin failed to block cell dissemination. Furthermore, we found that pronounced Akt1 signaling increased the susceptibility to cell apoptosis after treatments with 2-deoxy-D-glucose (2-DG) and lovastatin, enzymatic inhibitors of hexokinase, and 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase (HMGCR), especially in combination with radiotherapy and doxorubicin. In conclusion, these data suggest that restriction of glucose metabolism and the mevalonate pathway, in combination with standard therapy, may increase therapy success in RMS tumors characterized by a dysregulated Akt signaling. Full article

Methane (CH₄) from enteric fermentation accounts for 3 to 5% of global anthropogenic greenhouse gas emissions, which contribute to climate change. Cost-effective strategies are needed to reduce feed energy losses as enteric CH₄ while improving ruminant production efficiency. Mitigation strategies need to be environmentally friendly, easily adopted by producers and accepted by consumers. However, few sustainable CH₄ mitigation approaches are available. Recent studies show that the chemically synthesized CH₄ inhibitor 3-nitrooxypropanol is one of the most effective approaches for enteric CH₄ abatement. 3-nitrooxypropanol specifically targets the methyl-coenzyme M reductase and inhibits the final catalytic step in methanogenesis in rumen archaea. Providing 3-nitrooxypropanol to dairy and beef cattle in research studies has consistently decreased enteric CH₄ production by 30% on average, with reductions as high as 82% in some cases. Efficacy is positively related to 3-NOP dose and negatively affected by neutral detergent fiber concentration of the diet, with greater responses in dairy compared with beef cattle when compared at the same dose. This review collates the current literature on 3-nitrooxypropanol and examines the overall findings of meta-analyses and individual studies to provide a synthesis of science-based information on the use of 3-nitrooxypropanol for CH₄ abatement. The intent is to help guide commercial adoption at the farm level in the future. There is a significant body of peer-reviewed scientific literature to indicate that 3-nitrooxypropanol is effective and safe when incorporated into total mixed rations, but further research is required to fully understand the long-term effects and the interactions with other CH₄ mitigating compounds. Full article

Methane is a greenhouse gas which poses a great threat to life on earth as its emissions directly contribute to global warming and methane has a 28-fold higher warming potential over that of carbon dioxide. Ruminants have been identified as a major source of methane emission as a result of methanogenesis by their respective gut microbiomes. Various plants produce highly bioactive compounds which can be investigated to find a potential inhibitor of methyl-coenzyme M reductase (the target protein for methanogenesis). To speed up the process and to limit the use of laboratory resources, the present study uses an in-silico molecular docking approach to explore the anti-methanogenic properties of phytochemicals from Cymbopogon citratus, Origanum vulgare, Lavandula officinalis, Cinnamomum zeylanicum, Piper betle, Cuminum cyminum, Ocimum gratissimum, Salvia sclarea, Allium sativum, Rosmarinus officinalis and Thymus vulgaris. A total of 168 compounds from 11 plants were virtually screened. Finally, 25 scrutinized compounds were evaluated against methyl-coenzyme M reductase (MCR) protein using the AutoDock 4.0 program. In conclusion, the study identified 21 out of 25 compounds against inhibition of the MCR protein. Particularly, five compounds: rosmarinic acid (−10.71 kcal/mol), biotin (−9.38 kcal/mol), α-cadinol (−8.16 kcal/mol), (3R,3aS,6R,6aR)-3-(2H-1,3-benzodioxol-4-yl)-6-(2H-1,3-benzodioxol-5-yl)-hexahydrofuro[3,4-c]furan-1-one (−12.21 kcal/mol), and 2,4,7,9-tetramethyl-5decyn4,7diol (−9.02 kcal/mol) showed higher binding energy towards the MCR protein. In turn, these compounds have potential utility as rumen methanogenic inhibitors in the proposed methane inhibitor program. Ultimately, molecular dynamics simulations of rosmarinic acid and (3R,3aS,6R,6aR)-3-(2H-1,3-benzodioxol-4-yl)-6-(2H-1,3-benzodioxol-5-yl)-hexahydrofuro[3,4-c]furan-1-one yielded the best possible interaction and stability with the active site of 5A8K protein for 20 ns. Full article