Modulation of adenine phosphoribosyltransferase-mediated salvage pathway to accelerate diabetic wound healing.

Adenine phosphoribosyltransferase (APRT) is the key enzyme involved in purine salvage by the incorporation of adenine and phosphoribosyl pyrophosphate to provide adenylate nucleotides. To evaluate the role of APRT in the repair processes of cutaneous wounds in healthy skin and in diabetic patients, a diabetic mouse model (db/db) and age-matched wild-type mice were used. Moreover, the topical application of adenine was assessed. In vitro studies, analytical, histological, and immunohistochemical methods were used. Diabetic mice treated with adenine exhibited elevated ATP levels in organismic skin and accelerated wound healing. In vitro studies showed that APRT utilized adenine to rescue cellular ATP levels and proliferation from hydrogen peroxide-induced oxidative damage. HPLC-ESI-MS/MS-based analysis of total adenylate nucleotides in NIH-3T3 fibroblasts demonstrated that adenine addition enlarged the cellular adenylate pool, reduced the adenylate energy charge, and provided additional AMP for the further generation of ATP. These data indicate an upregulation of APRT in skin wounds, highlighting its role during the healing of diabetic wounds through regulation of the nucleotide pool after injury. Furthermore, topical adenine supplementation resulted in an enlargement of the adenylate pool needed for the generation of ATP, an important molecule for wound repair.

Development of an antigen-capture ELISA for beak and feather disease virus.

Psittacine beak and feather disease (PBFD) is characterised by degenerative feather, feather dystrophy, and beak deformity. Sometimes acute forms can lead to fatal cases in nestlings. The worldwide distribution of this disease affects numerous species of parrots with an average prevalence of 40%, including in Taiwan. The pathogen of PBFD is beak and feather disease virus (BFDV), which is a single-stranded circular DNA virus, circovirus. To date, hemagglutination and PCR assays have been routinely used to detect this virus. In this study, both the replication-associated protein (Rep) and the structural capsid protein (Cap) were expressed and then used as antigens for the production of monoclonal antibodies. Conserved epitopes recognised by the anti-Cap and anti-Rep monoclonal antibodies were determined to be NFEDYRI and LSALKKM, respectively. Clinical samples collected from different species of parrots were tested by hemagglutination, PCR, and anti-Cap antigen-capture ELISA assays and the positive rates were the same at 49%. Thus, this anti-Cap antigen-capture ELISA is able to be used for the rapid identification of BFDV-infected birds in a non-invasive manner.

The genotyping of infectious bronchitis virus in Taiwan by a multiplex amplification refractory mutation system reverse transcription polymerase chain reaction.

Infectious bronchitis virus (IBV; Avian coronavirus) causes acute respiratory and reproductive and urogenital diseases in chickens. Following sequence alignment of IBV strains, a combination of selective primer sets was designed to individually amplify the IBV wild-type and vaccine strains using a multiplex amplification refractory mutation system reverse transcription polymerase chain reaction (ARMS RT-PCR) approach. This system was shown to discriminate the IBV wild-type and vaccine strains. Moreover, an ARMS real-time RT-PCR (ARMS qRT-PCR) was combined with a high-resolution analysis (HRMA) to establish a melt curve analysis program. The specificity of the ARMS RT-PCR and the ARMS qRT-PCR was verified using unrelated avian viruses. Different melting temperatures and distinct normalized and shifted melting curve patterns for the IBV Mass, IBV H120, IBV TW-I, and IBV TW-II strains were detected. The new assays were used on samples of lung and trachea as well as virus from allantoic fluid and cell culture. In addition to being able to detect the presence of IBV vaccine and wild-type strains by ARMS RT-PCR, the IBV Mass, IBV H120, IBV TW-I, and IBV TW-II strains were distinguished using ARMS qRT-PCR by their melting temperatures and by HRMA. These approaches have acceptable sensitivities and specificities and therefore should be able to serve as options when carrying out differential diagnosis of IBV in Taiwan and China.

Development of a multiplex amplification refractory mutation system reverse transcription polymerase chain reaction assay for the differential diagnosis of Feline leukemia virus vaccine and wild strains.

A multiplex amplification refractory mutation system reverse transcription polymerase chain reaction (ARMS RT-PCR) was developed for the differential diagnosis of Feline leukemia virus (FeLV) vaccine and wild-type strains based on a point mutation between the vaccine strain (S) and the wild-type strain (T) located in the p27 gene. This system was further upgraded to obtain a real-time ARMS RT-PCR (ARMS qRT-PCR) with a high-resolution melt analysis (HRMA) platform. The genotyping of various strains of FeLV was determined by comparing the HRMA curves with the defined wild-type FeLV (strain TW1), and the results were expressed as a percentage confidence. The detection limits of ARMS RT-PCR and ARMS qRT-PCR combined with HRMA were 100 and 1 copies of transcribed FeLV RNA per 0.5 ml of sample, respectively. No false-positive results were obtained with 6 unrelated pathogens and 1 feline cell line. Twelve FeLV Taiwan strains were correctly identified using ARMS qRT-PCR combined with HRMA. The genotypes of the strains matched the defined FeLV wild-type strain genotype with at least 91.17% confidence. A higher degree of sequence polymorphism was found throughout the p27 gene compared with the long terminal repeat region. In conclusion, the current study describes the phylogenetic relationship of the FeLV Taiwan strains and demonstrates that the developed ARMS RT-PCR assay is able to be used to detect the replication of a vaccine strain that has not been properly inactivated, thus acting as a safety check for the quality of FeLV vaccines.

Differential diagnosis of Goatpox virus in Taiwan by multiplex polymerase chain reaction assay and high-resolution melt analysis.

The A32L gene from a Goatpox virus (GTPV) strain isolated from a goat in Yunlin County (Taiwan) displays several substitutions compared with the sequence of the Kenyan GTPV vaccine strain SGP0240 and the Pellor GTPV strain. Samples from the skin lesions on 6 goats with GTPV infection or from goats with Orf virus (ORFV) infection were tested in a multiplex polymerase chain reaction (PCR) system that used primers GPF, GPR1, and GPR2 as well as previously published primers specific for ORFV. These primers were able to amplify either GTPV or ORFV without cross-reactivity. A high-resolution melt analysis (HRMA) was carried out on amplified DNA from the skin lesions of 6 goats with GTPV infection and with the GTPV SGP0240 strain. The results indicated that the melting temperature profiles amplified from samples with Yunlin GTPV infection can be differentiated from the GTPV SGP0240 strain. The findings showed that a successful differential assay for these GTPVs had been developed. Accordingly, both methods can be used to detect and differentiate GTPV isolated from animals that may have either been vaccinated or been infected with a wild strain. The multiplex PCR and HRMA could be used on skin samples of suspected cases to serve as the front-line and confirmative assays, respectively, which will be beneficial to the eradication of GTPV.

Ketone bodies upregulate endothelial connexin 43 (Cx43) gap junctions.

Ketosis occurs as a metabolic consequence of negative energy balance in post-calving lactating dairy cows. Metabolism of free fatty acids, released from adipose tissue, generates excessive amounts of acetoacetate (AcAc), ß-hydroxybutyrate (BHB), and acetone (Ac) in the liver, which are released into the blood. The effects of ketone bodies on endothelial cells include increased rates of portal vein and liver blood flow and decreased cytokine secretion in response to both bacterial and viral infections. The aim of the current study was to understand the effects of AcAc, BHB and Ac, on expression of connexin 43 (Cx43) and gap junctional intercellular coupling (GJIC) in bovine aortic endothelial cells (BAECs). Confocal microscopy, Western blotting, and real-time quantitative RT-PCR indicated that Cx43 mRNA and protein expression increased after endothelial cell exposure to ketone bodies and that this was accompanied by upregulation of GJIC and cell migration. These effects were most obvious when BAECs were treated with a combination of the three ketones. Ketone bodies were shown to activate ERK and p38 MAPK as early as 3h after treatment and an ERK inhibitor (PD98059) or p38 MAPK inhibitor (SB203580) were found to antagonise the ketone-induced increase in Cx43 protein expression. Thus, ketone bodies up-regulate Cx43 expression and GJIC in BAECs via activation of ERK and p38 MAPK.