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Propionic acidemia/aciduria (PA) is an ultra-rare, life-threatening, inherited metabolic disorder caused by deficiency of the mitochondrial enzyme, propionyl-CoA carboxylase (PCC) composed of six alpha (PCCA) and six beta (PCCB) subunits. We herein report an enzyme replacement approach to treat PA using a combination of two messenger RNAs (mRNAs) (dual mRNAs) encoding both human PCCA (hPCCA) and PCCB (hPCCB) encapsulated in biodegradable lipid nanoparticles (LNPs) to produce functional PCC enzyme in liver. In patient fibroblasts, dual mRNAs encoded proteins localize in mitochondria and produce higher PCC enzyme activity vs. single (PCCA or PCCB) mRNA alone. In a hypomorphic murine model of PA, dual mRNAs normalize ammonia similarly to carglumic acid, a drug approved in Europe for the treatment of hyperammonemia due to PA. Dual mRNAs additionally restore functional PCC enzyme in liver and thus reduce primary disease-associated toxins in a dose-dependent manner in long-term 3- and 6-month repeat-dose studies in PA mice. Dual mRNAs are well-tolerated in these studies with no adverse findings. These studies demonstrate the potential of mRNA technology to chronically administer multiple mRNAs to produce large complex enzymes, with applicability to other genetic disorders. Propionic acidemia is a serious pediatric inherited disorder with no effective treatments. Here the authors demonstrate that delivering dual mRNAs as an enzyme replacement approach can be used as an effective therapy in a mouse model of propionic acidemia, with potential applicability to chronically administer multiple mRNAs in other genetic disorders.

Arginase deficiency is caused by biallelic mutations in arginase 1 (ARG1), the final step of the urea cycle, and results biochemically in hyperargininemia and the presence of guanidino compounds, while it is clinically notable for developmental delays, spastic diplegia, psychomotor function loss, and (uncommonly) death. There is currently no completely effective medical treatment available. While preclinical strategies have been demonstrated, disadvantages with viral-based episomal-expressing gene therapy vectors include the risk of insertional mutagenesis and limited efficacy due to hepatocellular division. Recent advances in messenger RNA (mRNA) codon optimization, synthesis, and encapsulation within biodegradable liver-targeted lipid nanoparticles (LNPs) have potentially enabled a new generation of safer, albeit temporary, treatments to restore liver metabolic function in patients with urea cycle disorders, including ARG1 deficiency. In this study, we applied such technologies to successfully treat an ARG1-deficient murine model. Mice were administered LNPs encapsulating human codon-optimized ARG1 mRNA every 3 d. Mice demonstrated 100% survival with no signs of hyperammonemia or weight loss to beyond 11 wk, compared with controls that perished by day 22. Plasma ammonia, arginine, and glutamine demonstrated good control without elevation of guanidinoacetic acid, a guanidino compound. Evidence of urea cycle activity restoration was demonstrated by the ability to fully metabolize an ammonium challenge and by achieving near-normal ureagenesis; liver arginase activity achieved 54% of wild type. Biochemical and microscopic data showed no evidence of hepatotoxicity. These results suggest that delivery of ARG1 mRNA by liver-targeted nanoparticles may be a viable gene-based therapeutic for the treatment of arginase deficiency.

Background Soybean is one of the most important oil crops in the world. The domestication of wild soybean has resulted in significant changes in the seed oil content and seed size of cultivated soybeans. To better understand the molecular mechanisms of seed formation and oil content accumulation, WDD01514 (E1), ZYD00463 (E2), and two extreme progenies (E23 and E171) derived from RILs were used for weighted gene coexpression network analysis (WGCNA) combined with transcriptome analysis. Results In this study, both seed weight and oil content in E1 and E171 were significantly higher than those in E2 and E23, and 20 DAF and 30 DAF may be key stages of soybean seed oil content accumulation and weight increase. Pathways such as “Photosynthesis”, “Carbon metabolism”, and “Fatty acid metabolism”, were involved in oil content accumulation and grain formation between wild and cultivated soybeans at 20 and 30 DAF according to RNA-seq analysis. A total of 121 oil content accumulation and 189 seed formation candidate genes were screened from differentially expressed genes. WGCNA identified six modules related to seed oil content and seed weight, and 76 candidate genes were screened from modules and network. Among them, 16 genes were used for qRT-PCR and tissue specific expression pattern analysis, and their expression-levels in 33-wild and 23-cultivated soybean varieties were subjected to correlation analysis; some key genes were verified as likely to be involved in oil content accumulation and grain formation. Conclusions Overall, these results contribute to an understanding of seed lipid metabolism and seed size during seed development, and identify potential functional genes for improving soybean yield and seed oil quantity.

Alpha 1-antitrypsin (AAT) deficiency arises from an inherited mutation in the SERPINA1 gene. The disease causes damage in the liver where the majority of the AAT protein is produced. Lack of functioning circulating AAT protein also causes uninhibited elastolytic activity in the lungs leading to AAT deficiency-related emphysema. The only therapy apart from liver transplantation is augmentation with human AAT protein pooled from sera, which is only reserved for patients with advanced lung disease caused by severe AAT deficiency. We tested modified mRNA encoding human AAT in primary human hepatocytes in culture, including hepatocytes from AAT deficient patients. Both expression and functional activity were investigated. Secreted AAT protein increased from 1,14 to 3,43 µg/ml in media from primary human hepatocytes following mRNA treatment as investigated by ELISA and western blot. The translated protein showed activity and protease inhibitory function as measured by elastase activity assay. Also, mRNA formulation in lipid nanoparticles was assessed for systemic delivery in both wild type mice and the NSG-PiZ transgenic mouse model of AAT deficiency. Systemic intravenous delivery of modified mRNA led to hepatic uptake and translation into a functioning protein in mice. These data support the use of systemic mRNA therapy as a potential treatment for AAT deficiency.

Archaeal and eukaryotic translation elongation factor 2 contain a unique post-translationally modified histidine residue called diphthamide, which is the target of diphtheria toxin. The biosynthesis of diphthamide was proposed to involve three steps, with the first being the formation of a C–C bond between the histidine residue and the 3-amino-3-carboxypropyl group of S -adenosyl- l -methionine (SAM). However, further details of the biosynthesis remain unknown. Here we present structural and biochemical evidence showing that the first step of diphthamide biosynthesis in the archaeon Pyrococcus horikoshii uses a novel iron–sulphur-cluster enzyme, Dph2. Dph2 is a homodimer and each of its monomers can bind a [4Fe–4S] cluster. Biochemical data suggest that unlike the enzymes in the radical SAM superfamily, Dph2 does not form the canonical 5′-deoxyadenosyl radical. Instead, it breaks the C γ,Met –S bond of SAM and generates a 3-amino-3-carboxypropyl radical. Our results suggest that P. horikoshii Dph2 represents a previously unknown, SAM-dependent, [4Fe–4S]-containing enzyme that catalyses unprecedented chemistry. First steps towards synthetic diphthamide Diphthamide, the target of diphtheria toxin, is a post-translationally modified histidine residue in archaeal and eukaryotic translation elongation factor 2 (EF2). It has been studied for more than three decades, but some aspects of its mechanism of biosynthesis remain elusive. The process is thought to involve three steps, the first being the formation of a C–C bond between the histidine residue and the 3-amino-3-carboxypropyl group of S -adenosyl- l -methionine (SAM). A study of the archaeon Pyrococcus horikoshii now shows that in this organism at least, the first step in diphthamide biosynthesis requires an unusual iron–sulphur cluster enzyme, Dph2. Biochemical data suggest that — in contrast to the mechanism in other radical SAM enzymes — Dph2 breaks the C γMet –S bond of the SAM cofactor. The enzyme then transfers the 3-amino-3-carboxylpropyl group to EF2, possibly via a radical mechanism. Translation elongation factor 2 (EF2) from archaea and eukaryotes contains a unique, post-translationally modified histidine residue called diphthamide, which is the target of diphtheria toxin. The biosynthesis of diphthamide involves three steps; here it is shown that the first step in the archaeon Pyrococcus horikoshii requires an unusual iron–sulphur-cluster enzyme, Dph2. It catalyses unprecedented chemistry.

Acute intermittent porphyria (AIP) results from haploinsufficiency of porphobilinogen deaminase (PBGD), the third enzyme in the heme biosynthesis pathway. Patients with AIP have neurovisceral attacks associated with increased hepatic heme demand. Phenobarbital-challenged mice with AIP recapitulate the biochemical and clinical characteristics of patients with AIP, including hepatic overproduction of the potentially neurotoxic porphyrin precursors. Here we show that intravenous administration of human PBGD (hPBGD) mRNA (encoded by the gene HMBS ) encapsulated in lipid nanoparticles induces dose-dependent protein expression in mouse hepatocytes, rapidly normalizing urine porphyrin precursor excretion in ongoing attacks. Furthermore, hPBGD mRNA protected against mitochondrial dysfunction, hypertension, pain and motor impairment. Repeat dosing in AIP mice showed sustained efficacy and therapeutic improvement without evidence of hepatotoxicity. Finally, multiple administrations to nonhuman primates confirmed safety and translatability. These data provide proof-of-concept for systemic hPBGD mRNA as a potential therapy for AIP. Systemic administration of human PBGD mRNA encapsulated in lipid nanoparticles ameliorates disease phenotypes in mouse and rabbit models of acute hepatic porphyria and is safe in nonhuman primates.

In order to reduce the health risks associated with red meat as listed by the World Health Organization, the work presented in this article aimed to elucidate the interaction between 5'-CMP-supplemented feed and N-glycolylneuraminic acid (Neu5Gc) in experimental animals in vivo. 5'-CMP was added to the diet of 90-, 180-, and 270-day-old Xiang pigs, and after 30 days, the Neu5Gc contents, physicochemical parameters, and free amino acid contents of muscle and internal viscera were measured by high-performance liquid chromatography coupled with fluorescence detection. The mechanism by which 5'-CMP affects Neu5Gc contents was investigated using molecular docking. Results show that 5'-CMP significantly decreased the Neu5Gc content in 180-day-old Xiang pigs (P < 0.05) but had no effect on the Neu5Gc contents in 90- and 270-day-old Xiang pigs. Umami amino acids were significantly increased in 180-day-old Xiang pigs. In the 90- and 270-day-old pigs, histidine increased by 10.38 and 17.87%, respectively. The other free amino acids were either reduced or not affected. Moreover, the 5'-CMP-supplemented diet did not affect the physicochemical parameters of the longissimus muscle in the Xiang pigs. 5'-CMP could occupy almost all the sialyltransferase active-site residues but not His302 and showed inhibition of the sialyltransferase activity. The results provided an experimental basis for the subsequent reduction of Neu5Gc in red meat before slaughter.

In order to reduce the health risks associated with red meat as listed by the World Health Organization (WHO), this paper aimed to elucidate the interaction between cytidine 5'-monophosphate (5'-CMP) supplemented feed and N-glycolylneuraminic acid (Neu5Gc) in experimental animals in vivo. 5'-CMP was added to the diet of 90, 180, and 270 day old Xiang pigs, and after 30 days, the Neu5Gc contents, physicochemical parameters, and free amino acid contents of muscle and internal viscera were measured by high performance liquid chromatography coupled with fluorescence detector (HPLC/FLD). The mechanism by which 5'-CMP affects Neu5Gc contents was investigated by using the molecular docking. The results show that 5'-CMP significantly decreased Neu5Gc content in 180 day old Xiang pigs (P < 0.05), but had no effect on the Neu5Gc contents in 90 and 270 day old Xiang pigs. Umami amino acids were significantly increased in 180 day old Xiang pigs. In the 90 and 270 day old pigs, histidine increased by 10.38% and 17.87%, respectively. The other free amino acids were either reduced or not affected. Moreover, the 5'-CMP diet did not affect the physicochemical parameters of the longissimus muscle in the Xiang pigs. The 5'-CMP could occupy almost all the ST active sites without HIS302 and show inhibition of the ST activity. The results provided an experimental basis for the subsequent reduction of Neu5Gc in red meat before slaughter.

Nature 465, 891–896 (2010) In this Article, the following studies, which led to the identification of diphthamide structure and its biosynthetic genes, should have been cited 1,2,3,4,5 . Reference 1 reports the presence of an unusual amino acid at the ADP-ribosylation site of elongation factor 2 (EF2); ref.

Archaeal and eukaryotic translation elongation factor 2 contain a unique posttranslationally modified histidine residue called “diphthamide”, the target of diphtheria toxin. The biosynthesis of diphthamide were proposed to involve three steps, with the first step being the formation of a C-C bond between the histidine residue and the 3-amino-3-carboxypropyl group of S-adenosylmethionine (SAM). However, details of the biosynthesis have remained unknown. Here we present structural and biochemical evidence showing that the first step of diphthamide biosynthesis in the archaeon Pyrococcus horikoshii uses a novel iron-sulfur cluster enzyme, Dph2. Dph2 is a homodimer and each monomer contains a [4Fe-4S] cluster. Biochemical data suggest that unlike the enzymes in the radical SAM superfamily, Dph2 does not form the canonical 5′-deoxyadenosyl radical. Instead, it breaks the Cγ,Met-S bond of SAM and generates a 3-amino-3-carboxylpropyl radical. This work suggests that Pyrococcus horikoshii Dph2 represents a novel SAM-dependent [4Fe-4S]-containing enzyme that catalyzes unprecedented chemistry.