Pigs lacking the SRCR5 domain of CD163 protein demonstrate heritable resistance to the PRRS virus and no changes in animal performance from birth to maturity.

Porcine reproductive and respiratory syndrome (PRRS) is one of the world's most persistent viral pig diseases, with a significant economic impact on the pig industry. PRRS affects pigs of all ages, causing late-term abortions and stillbirths in sows, respiratory disease in piglets, and increased susceptibility to secondary bacterial infection with a high mortality rate. PRRS disease is caused by a positive single-stranded RNA PRRS virus (PRRSV), which has a narrow host-cell tropism limited to monocyte-macrophage lineage cells. Several studies demonstrated that the removal of CD163 protein or, as a minimum, its scavenger receptor cysteine-rich domain 5 (SRCR5) precludes the viral genome release, conferring resistance to PRRSV in live animals. Today, very limited information exists about the impact of such edits on animal performance from birth to maturity in pigs. Using CRISPR-Cas9 with dual-guide RNAs and non-homologous end joining (NHEJ), first-generation (E0) pigs were produced with a deletion of exon 7 in the CD163 gene. The selected pigs were bred to produce the next three generations of pigs to establish multiple lines of pigs homozygous for the edited allele, thereby confirming that the CD163 gene with removed exon 7 was stable during multiple breeding cycles. The pigs were evaluated relative to non-edited pigs from birth to maturity, including any potential changes in meat composition and resistance to PRRSV. This study demonstrates that removing the SRCR5 domain from the CD163 protein confers resistance to PRRSV and, relative to unedited pigs, resulted in no detected differences in meat composition and no changes in the growth rate, health, and ability to farrow. Together, these results support the targeted use of gene editing in livestock animals to address significant diseases without adversely impacting the health and well-being of the animals or the food products derived from them.

An efficient process for synthesis of 2'-O-methyl and 3'-O-methyl guanosine from 2-aminoadenosine using diazomethane and the catalyst stannous chloride.

An improved strategy for the selective synthesis of 2'-O-methyl and 3'-O-methyl guanosine from 2-aminoadenosine is reported by using the catalyst stannous chloride. The regioselectivity of the 2' and 3'-O-alkylation was achieved by optimizing the addition, timing, and concentration of the catalysts and diazomethane during the methylation reaction. An efficient and selective alkylation at 2'-OH of 2-aminoadenosine was achieved by mixing a stoichiometric amount of stannous chloride at room temperature in DME The reaction mixture was stirred at 50 degrees C for 1 min and immediately followed by addition of diazomethane. The resulting 2'-O-methyl 2-aminoadenosine was treated with the enzyme adenosine deaminase, which resulted in an efficient conversion to the desired 2'-O-methylguanosine (98% yield). The product was isolated by crystallization. In contrast, the methylation at 3'-OH of 2-aminoadenosine was achieved by mixing a stoichiometric amount of stannous chloride in DMF and stirring at 50 degrees C for 15 min, followed by addition of diazomethane. The resulting mixture containing 3'-O-methyl-2-aminoadenosine in 90% yield and 2'-O-methyl-2-aminoadenosine in 10% yield was treated with the enzyme adenosine deaminase, which preferentially deaminated only 3'-O-methyl-2-aminoadenosine, resulting in the production of 3'-O-methylguanosine in 88% yield. Due to the extremely low solubility 3'-O-methylguanosine, the compound precipitated and was isolated by centrifugation. This synthetic route obviates the chromatographic purification. Selective monomethylation is achieved by using the unprotected ribonucleoside. As a result, the method described herein represents a significant improvement over the current synthetic approach by providing superior product yield and economy, a much more facile purification of 2',3'-O-methylated isomers, and eliminating the need for protected ribonucleosides reagents.

Oxidized lipids as mediators of coronary heart disease.

PURPOSE OF REVIEW: To summarize the recent evidence on the physiological relevance of the view that LDL lipid oxidation may play a major role in the inflammatory reaction that leads to or amplifies atherogenesis. Oxidation of LDL phospholipids containing arachidonic acid at the sn-2 position occurs when a critical concentration of 'seeding molecules' derived from the lipoxygenase pathway is reached in LDL. This generates a series of biologically active, oxidized phospholipids that mediate the cellular events seen in the developing fatty streak. RECENT FINDINGS: We have observed that LDL from mice that are genetically predisposed to diet-induced atherosclerosis is highly proinflammatory when the mice are maintained on an atherogenic diet, when they are injected with LDL-derived oxidized phospholipids, or once they are infected with influenza A virus. Patients with coronary atherosclerosis also had highly proinflammatory LDL, despite having normal blood lipid levels or normal plasma HDL levels. SUMMARY: We and others have hypothesized that HDL and LDL-derived oxidized phospholipids may be part of a system of nonspecific innate immunity. We therefore propose that determination of HDL capacity against LDL oxidation and the detection of proinflammatory HDL may be a useful marker of susceptibility to atherosclerosis.