Fong-Yuan Lin

Urine neutrophil gelatinase-associated lipocalin (NGAL) as a biomarker for acute canine kidney injury

BackgroundBiomarkers for the early prediction of canine acute kidney injury (AKI) are clinically important. Recently, neutrophil gelatinase-associated lipocalin (NGAL) was found to be a sensitive biomarker for the prediction of human AKI at a very early stage and the development of AKI after surgery. However, NGAL has not yet been studied with respect to dog kidney diseases. The application of NGAL canine AKI was investigated in this study.ResultsThe canine NGAL gene was successfully cloned and expressed. Polyclonal antibodies against canine NGAL were generated and used to develop an ELISA for measuring NGAL protein in serum and urine samples that were collected from 39 dogs at different time points after surgery.AKI was defined by the standard method, namely a serum creatinine increase of greater than or equal to 26.5 μmol/L from baseline within 48 h. At 12 h after surgery, compared to the group without AKI (12 dogs), the NGAL level in the urine of seven dogs with AKI was significantly increased (median 178.4 pg/mL vs. 88.0 pg/mL), and this difference was sustained to 72 h.ConclusionAs the increase in NGAL occurred much earlier than the increase in serum creatinine, urine NGAL seems to be able to serve as a sensitive and specific biomarker for the prediction of AKI in dogs.

Phylogenetic analysis of parapoxviruses and the C-terminal heterogeneity of viral ATPase proteins.

Two outbreaks of orf virus (a parapoxvirus) infection in goats found in Nantou and Taiping of central Taiwan were investigated. The nucleotide and the amino acid sequences of viral B2L, E3L and A32L genes in these two outbreaks were analyzed, and each of their phylogenetic trees were also constructed. In the A32L gene, an unexpected deletion of 24 nucleotides was found in the Taiping strain. The A32L gene can encode an ATPase and is supposed to be involved in virion DNA packaging. The 24 nucleotides correspond to 8 amino acids residues of the viral ATPase, which are located near the C-terminal region of the enzyme. Moreover, two copies of the RGD sequence at C-terminal region of ATPase were found in the Nantou strain. The 24-nucleotide difference in the A32L gene indicated that the Nantou strain and the Taiping strain were two separate strains, and it can be used in differential molecular diagnosis. Moreover, the C-terminal heterogeneity was found to be a general feature of the viral ATPase. Lastly, similar functional motifs of the ATPase and the Ras proto-oncoprotein (a GTPase) are discussed.

Neutrophil Gelatinase–Associated Lipocalin in Dogs with Naturally Occurring Renal Diseases

Background Neutrophil gelatinase–associated lipocalin (NGAL) is released from renal tubular cells after injury and serves in humans as a real‐time indicator of active kidney damage, including acute kidney injury (AKI) and chronic kidney disease (CKD). However, NGAL concentrations in dogs with naturally occurring AKI or CKD rarely have been explored in detail. Hypothesis/Objectives The goal of this study was to evaluate whether NGAL can serve as a useful biomarker in dogs with naturally occurring renal disease. Animals Client‐owned dogs with renal disease (57) and control dogs without any disease (12) were examined. Methods Serum NGAL (sNGAL) and urine NGAL (uNGAL) concentrations were measured in each animal by a newly developed ELISA system. Demographic, hematologic, and serum biochemical data were recorded. Survival attributable to AKI and CKD was evaluated at 30 days and 90 days, respectively. Results Serum and urine NGAL concentrations in azotemic dogs were significantly higher than in nonazotemic dogs and were highly correlated with serum creatinine concentration (P < .05). Among CKD dogs, death was associated with significantly higher sNGAL and uNGAL concentrations compared with survivors. Receiver‐operating characteristic curve (ROC) analysis showed that sNGAL was better than serum creatinine concentration when predicting clinical outcomes for CKD dogs (P < .05). The best cutoff point for sNGAL was 50.6 ng/mL, which gave a sensitivity and a specificity of 76.9 and 100%, respectively. Furthermore, dogs that had higher concentrations of sNGAL survived for a significantly shorter time. Conclusion sNGAL is a useful prognostic marker when evaluating dogs with CKD.

Purification and functional motifs of the recombinant ATPase of orf virus

Our previous study showed that the recombinant ATPase encoded by the A32L gene of orf virus displayed ATP hydrolysis activity as predicted from its amino acids sequence. This viral ATPase contains four known functional motifs (motifs I-IV) and a novel AYDG motif; they are essential for ATP hydrolysis reaction by binding ATP and magnesium ions. The motifs I and II correspond with the Walker A and B motifs of the typical ATPase, respectively. To examine the biochemical roles of these five conserved motifs, recombinant ATPases of five deletion mutants derived from the Taiping strain were expressed and purified. Their ATPase functions were assayed and compared with those of two wild type strains, Taiping and Nantou isolated in Taiwan. Our results showed that deletions at motifs I-III or IV exhibited lower activity than that of the wild type. Interestingly, deletion of AYDG motif decreased the ATPase activity more significantly than those of motifs I-IV deletions. Divalent ions such as magnesium and calcium were essential for ATPase activity. Moreover, our recombinant proteins of orf virus also demonstrated GTPase activity, though weaker than the original ATPase activity.

The Interplay of Reovirus with Autophagy

Autophagy participates in multiple fundamental physiological processes, including survival, differentiation, development, and cellular homeostasis. It eliminates cytoplasmic protein aggregates and damaged organelles by triggering a series of events: sequestering the protein substrates into double-membrane vesicles, fusing the vesicles with lysosomes, and then degrading the autophagic contents. This degradation pathway is also involved in various disorders, for instance, cancers and infectious diseases. This paper provides an overview of modulation of autophagy in the course of reovirus infection and also the interplay of autophagy and reovirus.

The different molecular forms of urine neutrophil gelatinase-associated lipocalin present in dogs with urinary diseases.

BackgroundNeutrophil gelatinase-associated lipocalin (NGAL) is a useful biomarker for the early prediction of renal diseases. NGAL may exist as monomer, dimer and/or NGAL/MMP-9 complex forms in humans. In this study, the existence of various forms of NGAL in urine (uNGAL) was determined and whether these forms are related to the different urinary diseases found in dogs is further discussed.ResultsEighty-one urine samples from dogs with different forms of renal disease (41), pyuria (19) and a number of non-renal related diseases (10), as well as healthy dogs (11), were collected. uNGAL concentrations and their molecular forms in dogs were measured by ELISA and Western blot analysis, respectively. The uNGAL concentrations of dogs with pyuria (median: 15.35 ng/mL) were significantly higher than those of the healthy control animals (median: 3.92 ng/mL) (p < 0.01), but lower than those of dogs with renal diseases (median: 23.77 ng/mL). Each NGAL molecular form could be detected in dog urine. In particular, monomer was detected more frequently in patients with renal disease than those with non-renal diseases; while the dimer form appeared in a significantly higher percentage of cases with pyuria compared to those without pyuria. The NGAL/MMP-9 complex was found to exist not only in the patients with cystitis, but also in the cases with renal injury.ConclusionDifferent molecular forms of uNGAL can indicate different origins of the urinary abnormalities. Determining the molecular forms of uNGAL present in diseased dogs may provide clinical workers with a tool that will help the early and more precise detection of different urinary diseases.

M-specific reverse transcription loop-mediated isothermal amplification for detection of pandemic (H1N1) 2009 virus

Eur J Clin Invest 2011; 41 (4): 434–441

Functional expression of the recombinant ATPase of orf virus

Nucleotide sequence analysis has indicated that the A32L gene of orf virus can encode an ATPase (Chan et al. in Gene 432:44–53, 2009). In this work, we cloned the A32L gene into a prokaryotic expression vector, and the recombinant protein was expressed and purified. The antigenicity of recombinant ATPase was examined by immunoblotting, and its identity was confirmed by mass spectrometry. The ATP hydrolysis function of the purified recombinant protein was examined, and our results showed that it exhibited the ATPase activity. Similar to other viral ATPases, the ATPase of orf virus remained active in the presence of different divalent ions; nevertheless, unlike other viral ATPases, our recombinant ATPase exhibited similar enzymatic activity in reaction buffers of different pH.

The pseudorabies virus vhs protein cleaves RNA containing an IRES sequence

The virion host shutoff protein (vhs), encoded by the gene UL41, has RNase activity and is the key regulator of the early host shutoff response induced by type 1 herpes simplex virus. Despite low amino acid similarity, the vhs protein of the swine herpesvirus, pseudorabies virus (PrV), also exhibits RNase activity. However, the mechanism underlying the action of vhs remains undefined. Here, we report that the RNA degradation profile of PrV vhs is similar, but not identical, to that of type 1 herpes simplex virus vhs. Notably, the presence of a cap structure enhances both the degradation rate and the preferential targeting of the vhs protein towards the 3′‐end of the encephalomyocarditis virus internal ribosome entry site (IRES). Furthermore, type 1 herpes simplex virus vhs produces a simple degradation pattern, but PrV vhs gives rise to multiple intermediates. The results of northern blotting using probes recognizing various regions of the RNA substrate found that PrV vhs also cleaves downstream of the IRES region and this vhs protein overall shows 5′ to 3′ RNase activity. Moreover, addition of the translation initiation factors eIF4H and eIF4B significantly increased the RNase activity of recombinant PrV vhs against capped RNA. Nonetheless, these proteins did not fully reconstitute the IRES‐directed targeting pattern observed for vhs translated in a rabbit reticular lysate system. The interaction between PrV vhs and eIF4H/eIF4B implies that the translation initiation machinery within the cell is able to stimulate the nuclease activity of PrV vhs. However, this process remains inefficient in terms of the IRES‐targeting pattern.

Functional analysis of the short isoform of orf virus protein OV20.0

ABSTRACT Orf virus (ORFV) OV20.0L is an ortholog of vaccinia virus (VACV) gene E3L. The function of VACV E3 protein as a virulence factor is well studied, but OV20.0 has received less attention. Here we show that like VACV E3L, OV20.0L encodes two proteins, a full-length protein and a shorter form (sh20). The shorter sh20 is an N-terminally truncated OV20.0 isoform generated when a downstream AUG codon is used for initiating translation. These isoforms differed in cellular localization, with full-length OV20.0 and sh20 found throughout the cell and predominantly in the cytoplasm, respectively. Nonetheless, both OV20.0 isoforms were able to bind double-stranded RNA (dsRNA)-activated protein kinase (PKR) and dsRNA. Moreover, both isoforms strongly inhibited PKR activation as shown by decreased phosphorylation of the translation initiation factor eIF2α subunit and protection of Sindbis virus infection against the activity of interferon (IFN). In spite of this apparent conservation of function in vitro, a recombinant ORFV that was able to express only the sh20 isoform was attenuated in a mouse model. IMPORTANCE The OV20.0 protein of orf virus (ORFV) has two isoforms and contributes to virulence, but the roles of the two forms are not known. This study shows that the shorter isoform (sh20) arises due to use of a downstream initiation codon and is amino-terminally truncated. The sh20 form also differs in expression kinetics and cellular localization from full-length OV20.0. Similar to the full-length isoform, sh20 is able to bind dsRNA and PKR, inactivate PKR, and thus act as an antagonist of the interferon response in vitro. In vivo, however, wild-type OV20.0 could not be replaced with sh20 alone without a loss of virulence, suggesting that the functions of the isoforms are not simply redundant.