Metagenomics and Metabolomics Study on Microbial Diversity and Enzymatic Activities Changes on Cow Dung Based Low-Temperature Biogas Production by Entererococcus spp

To fill the gap between the rise in demand for energy and decline in the traditional energy sources such as coal, natural gas and nuclear energy, other alternative sources such as biogas are necessary. Studies have shown that the existing conditions within the fermentation realm control the microbial characteristics in biogas production. However, there is inadequate insight between the duration of fermentation and the microbial diversity, and with specific emphasis to cow manure as the substrate under low temperature fermentation. This study aimed at providing additional insight on the effect of varying fermentation duration (0 to 60 days) on the composition of the dominant microbial flora on cow dung based low-temperature (15 °C) biogas fermentation using metagenomics and metabolomics analyses approach. The study results showed that the main dominant community in the process of methanogenesis are the Spirochaetae, Synergistetes and Chloroflexi, and are new flora in the methane phase. In the peak stage of low-temperature biogas fermentation, the dominant bacteria groups were Methanosarcina and after adding 10% concentration of L1 bacteria. The prediction of metabolic pathway was mainly carbohydrate metabolism and amino acid metabolism with succinyl-CoA synthase a subunit, lactaldehyde reductase and the glutamate-glyoxylate aminotransferase being the main unique enzymes. The study therefore supports the potential of involving the reported dominant microbial communities and related enzymatic activities for improved biogas production under low temperature conditions.

Overexpression of Alanine-glyoxylate aminotransferase 2 Protects from Asymmetric Dimethylarginine-induced Endothelial Dysfunction and Aortic Remodeling

Objective: Elevated plasma concentrations of asymmetric dimethylarginine (ADMA) are associated with an increased risk of mortality and adverse cardiovascular outcomes. ADMA can be metabolized by dimethylarginine dimethylaminohydrolases (DDAHs) and by alanine-glyoxylate aminotransferase 2 (AGXT2). Deletion of DDAH1 in mice leads to elevation of ADMA in plasma and blood pressure, while overexpression of human DDAH1 is associated with a lower plasma ADMA concentration and protective cardiovascular effects. The possible role of alternative metabolism of ADMA by AGXT2 remains to be elucidated. The goal of the current study was to test the hypothesis that transgenic overexpression of AGXT2 leads to lowering of plasma levels of ADMA and protection from vascular damage in the setting of DDAH1 deficiency.Approach and Results: We generated transgenic mice (TG) with ubiquitous overexpression of AGXT2. qPCR and Western Blot confirmed the expression of the transgene. Systemic ADMA levels were decreased by 15% in TG mice. In comparison with wild type animals plasma levels of asymmetric dimethylguanidino valeric acid (ADGV), the AGXT2 associated metabolite of ADMA, were six times higher. We crossed AGXT2 TG mice with DDAH1 knockout mice and observed that upregulation of AGXT2 lowers plasma ADMA and pulse pressure and protects the mice from endothelial dysfunction and adverse aortic remodeling.Conclusions: Upregulation of AGXT2 led to lowering of ADMA levels and protection from ADMA-induced vascular damage in the setting of DDAH1 deficiency. This is especially important, because all the efforts to develop pharmacological ADMA-lowering interventions by means of upregulation of DDAHs have been unsuccessful.

Generation and characterization of a novel rat model of primary hyperoxaluria type 1 with a nonsense mutation in alanine-glyoxylate aminotransferase gene

Primary hyperoxaluria type 1 (PH1) is a severe inherited disorder caused by a genetic defect in alanine-glyoxylate aminotransferase (AGXT) that results in recurrent urolithiasis and renal failure. Animal models that precisely reflect the human PH1 phenotypes are lacking. We aimed to develop a novel PH1 rat model and study the mechanisms involved in PH1 deterioration. One-cell stage Sprague-Dawley embryos were injected with the CRISPR/Cas9 system to introduce a Q84X mutation in Agxt. Liver tissues were harvested for determining Agxt expression. Urine oxalate, crystals, and electrolyte levels in AgxtQ84X and wild-type (WT) littermates were evaluated. Kidney tissues were used for Pizzolato staining and kidney injury evaluation. Data showed that Agxt mRNA and protein were absent in AgxtQ84X rats. At 4 and 24 weeks, the AgxtQ84X rats displayed 2.3- and 3-fold higher urinary oxalate levels, respectively, compared with WT littermates. As a result, calcium oxalate (CaOx) crystals in urine were revealed in all AgxtQ84X rats, but none of the WT rats. We also observed bladder stones in 36.4% of the AgxtQ84X rats, of which 44.4% had renal CaOx deposition. Moreover, the elevated serum urea and creatinine levels indicated the impaired renal function in AgxtQ84X rats. Further investigation revealed significantly increased expression of inflammation-, necroptosis-, and fibrosis-related genes in the kidneys of AgxtQ84X rats with spontaneous CaOx deposition, indicating that these pathways are involved in PH1 deterioration. Collectively, these results suggest that this rat model has broad applicability in mechanistic studies and innovative therapeutics development for PH1 and other kidney stone diseases.

Diet and Adaptive Evolution of Alanine-Glyoxylate Aminotransferase Mitochondrial Targeting in Birds

Adaptations to different diets represent a hallmark of animal diversity. The diets of birds are highly variable, making them an excellent model system for studying adaptive evolution driven by dietary changes. To test whether molecular adaptations to diet have occurred during the evolution of birds, we examined a dietary enzyme alanine-glyoxylate aminotransferase (AGT), which tends to target mitochondria in carnivorous mammals, peroxisomes in herbivorous mammals, and both mitochondria and peroxisomes in omnivorous mammals. A total of 31 bird species were examined in this study, which included representatives of most major avian lineages. Of these, 29 have an intact mitochondrial targeting sequence (MTS) of AGT. This finding is in stark contrast to mammals, which showed a number of independent losses of the MTS. Our cell-based functional assays revealed that the efficiency of AGT mitochondrial targeting was greatly reduced in unrelated lineages of granivorous birds, yet it tended to be high in insectivorous and carnivorous lineages. Furthermore, we found that proportions of animal tissue in avian diets were positively correlated with mitochondrial targeting efficiencies that were experimentally determined, but not with those that were computationally predicted. Adaptive evolution of AGT mitochondrial targeting in birds was further supported by the detection of positive selection on MTS regions. Our study contributes to the understanding of how diet drives molecular adaptations in animals, and suggests that caution must be taken when computationally predicting protein subcellular targeting.

Metabolism of Oxalate in Humans: A Potential Role Kynurenine Aminotransferase/Glutamine Transaminase/Cysteine Conjugate Betalyase Plays in Hyperoxaluria

Hyperoxaluria, excessive urinary oxalate excretion, is a significant health problem worldwide. Disrupted oxalate metabolism has been implicated in hyperoxaluria and accordingly, an enzymatic disturbance in oxalate biosynthesis can result in the primary hyperoxaluria. Alanine-glyoxylate aminotransferase-1 and glyoxylate reductase, the enzymes involving glyoxylate (precursor for oxalate) metabolism, have been related to primary hyperoxalurias. Some studies suggest that other enzymes such as glycolate oxidase and alanine-glyoxylate aminotransferase-2 might be associated with primary hyperoxaluria as well, but evidence of a definitive link is not strong between the clinical cases and gene mutations. There are still some idiopathic hyperoxalurias, which require a further study for the etiologies. Some aminotransferases, particularly kynurenine aminotransferases, can convert glyoxylate to glycine. Based on biochemical and structural characteristics, expression level, and subcellular localization of some aminotransferases, a number of them appear able to catalyze the transamination of glyoxylate to glycine more efficiently than alanine glyoxylate aminotransferase-1. The aim of this minireview is to explore other undermining causes of primary hyperoxaluria and stimulate research toward achieving a comprehensive understanding of underlying mechanisms leading to the disease. Herein, we reviewed all aminotransferases in the liver for their functions in glyoxylate metabolism. Particularly, kynurenine aminotransferase-I and III were carefully discussed regarding their biochemical and structural characteristics, cellular localization, and enzyme inhibition. Kynurenine aminotransferase-III is, so far, the most efficient putative mitochondrial enzyme to transaminate glyoxylate to glycine in mammalian livers, which might be an interesting enzyme to look for in hyperoxaluria etiology of primary hyperoxaluria and should be carefully investigated for its involvement in oxalate metabolism.

The Second Life of Methylarginines as Cardiovascular Targets

Endogenous methylarginines were proposed as cardiovascular risk factors more than two decades ago, however, so far, this knowledge has not led to the development of novel therapeutic approaches. The initial studies were primarily focused on the endogenous inhibitors of nitric oxide synthases asymmetric dimethylarginine (ADMA) and monomethylarginine (MMA) and the main enzyme regulating their clearance dimethylarginine dimethylaminohydrolase 1 (DDAH1). To date, all the screens for DDAH1 activators performed with the purified recombinant DDAH1 enzyme have not yielded any promising hits, which is probably the main reason why interest towards this research field has started to fade. The relative contribution of the second DDAH isoenzyme DDAH2 towards ADMA and MMA clearance is still a matter of controversy. ADMA, MMA and symmetric dimethylarginine (SDMA) are also metabolized by alanine: glyoxylate aminotransferase 2 (AGXT2), however, in addition to methylarginines, this enzyme also has several cardiovascular protective substrates, so the net effect of possible therapeutic targeting of AGXT2 is currently unclear. Recent studies on regulation and functions of the enzymes metabolizing methylarginines have given a second life to this research direction. Our review discusses the latest discoveries and controversies in the field and proposes novel directions for targeting methylarginines in clinical settings.