Molecular epidemiology of bovine toroviruses circulating in South Korea

The prevalence of the bovine torovirus (BToV) and its genetic characterization have been reported in North America, Europe and Japan. Therefore, this study examined the prevalence and genetic diversity of the BToV in a total of 645 diarrheic fecal samples from 629 Korean native beef calf herds using RT-PCR and nested PCR with the primer pairs specific to a part of the BToV membrane (M) gene. Overall, 19 (2.9%) out of 645 diarrheic samples from 19 herds (6.9%) tested positive for BToVs by either RT-PCR or nested PCR. A comparison of the nucleotide (nt) and amino acid (aa) sequences of a part of the BToV M gene (409bp) among the BToVs showed the Korean BToVs to have comparatively higher sequence homology to the Japanese and Dutch BToVs than to the American and Italian BToVs. Generally, the Korean BToV strains clustered with the Japanese and Dutch BToV strains. However, the American and Italian BToV strains clustered on a separate major branch, suggesting that these are more distantly related to other known BToV strains. These results suggest that the BToV infections are sporadic in diarrheic calves in South Korea, and the Korean BToV strains are more closely related to the Japanese and Dutch BToVs than to the American and Italian BToVs.     

Dexamethasone induces apoptosis in proliferative canine tendon cells and chondrocytes

Dexamethasone (Dexa) has been commonly used in humans and domestic animals, particularly in the treatment of tendon injuries and cartilage degeneration. However, it is often associated with tendon rupture and impaired tendon and cartilage healing. In the present study, we investigated Dexa's in vitro effects on the growth of cell proliferation and the induction of apoptosis in canine Achilles tendon cells and chondrocytes. Cell proliferation after treatment with Dexa for two to six days was quantified by a 2,3-bis{2-methoxy-4-nitro-5-sulfophenyl}-2H-tetrazolium-5-carboxyanilide inner salt assay (XTT). The results showed that Dexa could inhibit the proliferation of tendon cells and chondrocytes at increasing concentrations (0.1-50 microg/ml) compared with untreated cells. Cell apoptosis was induced by Dexa, as evidenced by the typical nuclear apoptosis using Hoechst 33258 staining. Dexa increased the apoptosis of canine tendon cells and chondrocytes in a time-dependent manner. In canine tendon cells and chondrocytes that were treated with 25 and 50 microg/ml concentration of Dexa, the number of condensed apoptotic nuclei was significantly increased. In addition, culturing with Dexa and the glucocorticoid receptor blocker, mifepristone, significantly arrested apoptosis of tendon cells and chondrocytes. Based on our in vitro data, we hypothesized that in vivo treatment with glucocorticoids may diminish the proliferation of tendon and cartilage cells by increasing apoptosis and suppressing the proliferation. Our findings suggest that Dexa could be used with caution in dogs with articular or tendon problems.     

Transient increases of glutamic acid decarboxylase 67 immunoreactivity and its protein levels in the somatosensory cortex after transient cerebral ischemia in gerbils

In this study, we investigated changes in glutamic acid decarboxylase 67 (GAD67) immunoreactivity and its protein levels in the gerbil somatosensory cortex after ischemia/reperfusion. GAD67 immunoreactivity was significantly increased in layers III and V of the somatosensory cortex 12 hr after ischemia/reperfusion. Thereafter, GAD67 immunoreactivity was decreased with time after ischemia/reperfusion. GAD67 immunoreactivity in the somatosensory cortex 4 days after ischemia/reperfusion was similar to that in the sham-operated group. In addition, GAD67 protein levels were also significantly increased 12 hr after transient forebrain ischemia. These results suggest that the transient increase of GAD67 immunoreactivity in layers III and V may be associated with responses to transient ischemia-induced neuronal damage.     

Time-course changes in the expression levels of miR-122, -155, and -21 as markers of liver cell damage,inflammation, and regeneration in acetaminophen-induced liver injury in rats

Drug-induced liver injury (DILI) is a significant threat to patient health and a major concern during drug development. Recently, multiple circulating microRNAs (miRNAs) have been reported to be potential biomarkers for DILI. To adapt and validate miRNAs for clinical use, we investigated the time-course changes in miR-122 expression levels in an acetaminophen-induced liver injury model in rats. In addition, miR-155 and miR-21 were evaluated as makers of inflammation and regeneration, respectively, to characterize liver status. Our results revealed that miR-122 is an early and sensitive biomarker of hepatocellular injury at a stage when alanine transaminase, aspartate transaminase, and total bilirubin were not detectable. However, no significant differences in the expression levels of other miRNAs (miR-155 and -21) were observed between treatment and vehicle groups. Collectively, these time-course changes in the expression levels of miRNAs may be useful as markers for clinical decision-making, in the diagnosis and treatment of DILI.     

Ahmed glaucoma valve implantation with Ologen® Collagen Matrix for the surgical treatment of feline glaucoma

An Ahmed valve implantation with an Ologen^® Collagen Matrix (Ologen^® CM, Aeon Astron, Leiden, the Netherlands) was performed for the treatment of uncontrolled glaucoma in a cat. This cat was a 5‐year‐old castrated Russian Blue male with a 12‐week history of conjunctival hyperemia and mydriasis of the left eye. During the ophthalmic examination, the intraocular pressure (IOP) oculus sinister (OS) was 52 mmHg, and a narrow iridocorneal angle (ICA) was detected by gonioscopy. Medical treatment with Cosopt^® (2% dorzolamide and 0.5% timolol) failed to decrease the IOP. The left eye still had vision, and an Ahmed valve implantation was performed. During the gonioimplantation, Ologen^® CM was used to inhibit scar formation around the valve. Following the operation, the IOP was stable at an approximate average of 15 mmHg during the 7‐month follow‐up period, and vision in the left eye was retained without medication. An adequate subconjunctival filtering bleb was formed after 140 days. This is the first case report in which an Ahmed valve gonioimplant with an Ologen^® CM has been used for the surgical treatment of glaucoma in a cat.     

Expression of the glutamine metabolism‐related proteins glutaminase 1 and glutamate dehydrogenase in canine mammary tumours

Glutamine metabolism is an important metabolic pathway for cancer cell survival, and there is a critical connection between tumour growth and glutamine metabolism. Because of their similarities, canine mammary carcinomas are useful for studying human breast cancer. Accordingly, we investigated the correlations between the expression of glutamine metabolism‐related proteins and the pathological features of canine mammary tumours. We performed immunohistochemical and western blot analysis of 39 mammary tumour tissues. In immunohistochemical analysis, the expression of glutaminase 1 (GLS1) in the epithelial region increased according to the histological grade (P < .005). In the stromal region, complex‐type tumours displayed significantly higher GLS1 intensity than simple‐type tumours. However, glutamate dehydrogenase expression did not show the same tendencies as GLS1. The western blot results were consistent with the immunohistochemical findings. These results suggest that the expression of GLS1 is correlates with clinicopathological factors in canine mammary tumours and shows a similar pattern to human breast cancer.     

Integrin heterodimer α9β1 is localized on the surface of porcine spermatogonial stem cells in the undifferentiated state

A previous study found that undifferentiated porcine spermatogonial stem cells (SSCs) did not adhere to tenascin C, indicating that the integrin α₉ and β₁ subunits are inactive on the surface of porcine SSCs. However, that study used recombinant tenascin C without FNIII‐like repeats. Therefore, this study re‐evaluated the existence of integrin α₉β₁ actively functioning on the plasma membrane of porcine SSCs using full‐length native tenascin C with FNIII‐like repeats. The localization and function of the integrin heterodimer were confirmed using immunocytochemistry, attachment and antibody inhibition assays. In undifferentiated porcine SSCs with integrin α₉β₁ on the cell surface, adhesion to native tenascin C was significantly higher compared with cells lacking native tenascin C and functional blocking of integrin α₉β₁ significantly inhibited the attachment to native tenascin C compared with no functional blocking. Accordingly, we confirmed that the integrin α₉ and β₁ subunits function as an active heterodimer on the surface of porcine SSCs in the undifferentiated state.     

Sequential disruption of ALV host receptor genes reveals no sharing of receptors between ALV subgroups A,B, and J

Background: Previously, we showed that targeted disruption of viral receptor genes in avian leukosis virus(ALV)subgroups using clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR-associated protein 9(Cas9))-based genome editing confers resistance to ALV subgroups B and J. Here, we used the same strategy to target the receptor expressed by ALV subgroup A(TVA) and generate chicken cells resistant to infection by this virus.Results: CRISPR/Cas9-based disruption of exon 2 within the tva gene of DF-1 fibroblasts conferred resistance to infection by ALV subgroup A regardless of whether frameshift mutations were introduced during editing. Conversely,overexpression of the wild-type TVA receptor(wtTVA) by tva-modified DF-1 clones restored susceptibility to ALV subgroup A. The results confirm that exon 2, which contains the low-density lipoprotein receptor class A domain of TVA, is critical for virus entry. Furthermore, we sequentially modified DF-1 cells by editing the tva, tvb, and Na~+/H~+ exchange 1(chNHE1) genes, which are the specific receptors for ALV subgroups A, B, and J, respectively.Conclusions: Simultaneous editing of multiple receptors to block infection by different subgroups of ALV confirmed that ALV subgroups A, B, and J do not share host receptors. This strategy could be used to generate cells resistant to multiple viral pathogens that use distinct receptors for cell entry.     

Primordial germ cell-mediated transgenesis and genome editing in birds

Transgenesis and genome editing in birds are based on a unique germline transmission system using primordial germ cells(PGCs), which is quite different from the mammalian transgenic and genome editing system. PGCs are progenitor cells of gametes that can deliver genetic information to the next generation. Since avian PGCs were first discovered in nineteenth century, there have been numerous efforts to reveal their origin, specification, and unique migration pattern, and to improve germline transmission efficiency. Recent advances in the isolation and in vitro culture of avian PGCs with genetic manipulation and genome editing tools enable the development of valuable avian models that were unavailable before. However, many challenges remain in the production of transgenic and genome-edited birds,including the precise control of germline transmission, introduction of exogenous genes, and genome editing in PGCs.Therefore, establishing reliable germline-competent PGCs and applying precise genome editing systems are critical current issues in the production of avian models. Here, we introduce a historical overview of avian PGCs and their application, including improved techniques and methodologies in the production of transgenic and genome-edited birds, and we discuss the future potential applications of transgenic and genome-edited birds to provide opportunities and benefits for humans.