Treatment of contaminated radial fracture in Sprague-Dawley rats by application of a degradable polymer releasing gentamicin

Fracture-related infections remain a leading cause of morbidity and mortality. We aimed to establish a simple contaminated radial osteotomy model to assess the efficacy of a biodegradable polymer poly(sebacic-co-ricinoleic acid) [p(SA-RA)] containing 20% w/w gentamicin. A unilateral transverse osteotomy was induced in Sprague-Dawley (SD) rats, followed by application of Staphylococcus aureus suspension over the fracture. After successfully establishing the contaminated open fracture model, we treated the rats either systemically (intraperitoneal cefuroxime), locally with p(SA-RA) containing gentamicin, or both. Control groups included non-contaminated group and contaminated groups that were either untreated or treated with the polymer alone. After 4 weeks, the bones were subjected to micro-CT scanning and microbiological and histopathology evaluations. Micro-CT analysis revealed similar changes in the group subjected to both local and systemic treatment as in the non-contaminated control group. Lack of detectable bacterial growth was noted in most animals of the group subjected to both local and systemic treatment, and all samples were negative for S. aureus. Histopathological evaluation revealed that all treatment modalities containing antibiotics were highly effective in reducing infection and promoting callus repair, resulting in early bone healing. While p(SA-RA) containing gentamicin treatment showed better results than cefuroxime, the combination of local and systemic treatment displayed the highest therapeutic potential in this model.     

Carrier rate of the c.235G>C mutation in the bovine isoleucyl-tRNA synthetase (IARS) gene of Japanese Black cows at Kagoshima prefecture, Japan,and analysis of the metabolic profiling and reproductive performance of heterozygous cows

Bovine isoleucyl-tRNA synthetase (IARS) disorder, a major cause of weak calf syndrome, is caused by a homozygous missense (c.235G>C) mutation in the bovine IARS gene of Japanese Black (JB) cattle, which was identified in 2013. However, the extent to which the carrier rate has changed at Kagoshima prefecture, Japan, and whether the carrier status is associated with any clinical or reproductive problems, have yet to be ascertained. In this study, using a real-time polymerase chain reaction-based genotyping assay, we determined the carrier rate in a regional JB cow population at Kagoshima prefecture. Comparative analyses were performed on the metabolic profile test (MPT) results and reproductive performance data obtained for heterozygous carrier and homozygous wild-type cows. In 2009 and 2018, DNA samples were collected from 130 and 462 clinically healthy JB cows, respectively, in Kagoshima prefecture. MPT results and reproductive performance data were evaluated for 62 cows, comprising four heterozygous carriers and 58 wild-type cows. Genotyping revealed that the carrier rate was 6.9% in 2009 and 1.5% in 2018, the difference of which was statistically significant (P<0.005). There were no statistically significant differences between the carrier and wild-type cows with respect to either MPT results or reproductive performance, indicating that the carrier cows have necessary IARS activity to maintain minimal health and reproductive potential.     

Molecular characterization of Ascaridia galli from Bangladesh and development of a PCR method for distinguishing A. galli from Heterakis spp.

We analyzed the nuclear ribosomal internal transcribed spacer (ITS) 1 and ITS2 sequences for Bangladesh isolates of Ascaridia galli, and we determined that the sequences were unreliable as molecular markers for distinguishing A. galli from other Ascaridia species, because the sequences showed high identity with that of A. columbae. However, the ITS1 sequences were available for designing PCR primers distinguishable between Ascaridia galli and Heterakis spp. Bangladesh isolates of A. galli constituted a monophyletic clade along with other geographical isolates in the cytochrome c oxidase subunit I (COI) phylogenetic tree, however, we could not clarify the phylogenetic relationships between A. galli and other Ascaridia spp., because their available sequences in GenBank were very few. The developed PCR method using DNA from A. galli and Heterakis spp. eggs would enable differential diagnosis of the individual infections in the future.     

Relationship between anti-insulin antibody production and severe insulin resistance in a diabetic cat

A 5-year-old castrated male domestic shorthair cat was diagnosed with diabetic ketoacidosis and severe insulin resistance. Although the conventional treatment for diabetic ketoacidosis was provided, the cat required frequent hospitalization because of severe dehydration and repeated diabetic ketoacidosis. We detected anti-insulin antibodies for human in this cat. Serum insulin-binding IgG levels were markedly elevated compared with those in healthy cats and other diabetic cats. We initiated prednisolone to suppress the effects of anti-insulin antibodies. After initiation of prednisolone, the cat was gradually recovered with increasing activity and appetite. Furthermore, satisfactory glycemic control was achieved with combined subcutaneous injection of insulin detemir and insulin degludec.     

伪狂犬病病毒在NF-κB家族p65基因敲除细胞系的复制规律研究

试验旨在研究伪狂犬病病毒(PRV)在NF-κB家族p65基因敲除细胞系中的复制规律。利用慢病毒介导的CRISPR/Cas9基因定点修饰技术构建猪肺泡巨噬细胞(3D4/21)p65基因稳定敲除细胞系。通过构建p65-sgRNA重组质粒,转染至HEK293T/17细胞,收取慢病毒,感染3D4/21细胞后利用嘌呤霉素筛选获得多克隆细胞系,T7核酸酶检测敲除效率,再通过有限稀释法获得3D4/21-p65^-/-的稳定细胞系。CCK-8试剂盒检测3D4/21细胞中敲除p65基因后对细胞增殖的影响;流式细胞术检测PRV-GFP感染3D4/21及3D4/21-p65^-/-细胞后病毒增殖的差异;实时定量PCR检测PRV感染3D4/21及3D4/21-p65^-/-细胞后PRV gB、TK基因mRNA表达水平及PRV感染细胞诱导的IL-1β和IL-6基因mRNA水平表达的变化;Western blotting检测PRV-QXX感染3D4/21及3D4/21-p65^-/-细胞后PRV gB、gE蛋白的表达;滴度测定检测PRV-QXX感染3D4/21及3D4/21-p65^-/-细胞后子代病毒滴度。结果表明,sgRNA2和sgRNA3的基因编辑效率较高,对其进行克隆化培养进而获得敲除p65基因的稳定表达细胞系;CCK-8试剂盒检测细胞活力表明,p65基因敲除对细胞活力无影响;流式细胞仪检测表明,同一时间点PRV-GFP在3D4/21-p65^-/-中的增殖显著高于对照细胞;实时荧光定量PCR表明在3D4/21细胞中敲除p65基因促进了PRV gB、TK基因的mRNA表达水平,而抑制了IL-1β、IL-6基因的mRNA表达;Western blotting结果表明,在3D4/21细胞中敲除p65基因促进了PRV gB、gE蛋白的表达;滴度测定结果表明,同一时间点PRV-QXX在3D4/21-p65^-/-细胞中子代病毒的复制显著高于对照细胞。以上结果均表明,p65基因敲除可促进PRV在3D4/21细胞中复制。     

The thermal antinociceptive effects of a high-concentration formulation of buprenorphine alone or followed by hydromorphone in conscious cats

ObjectiveTo evaluate the thermal antinociceptive effects of a high-concentration formulation of buprenorphine alone or followed by hydromorphone in conscious cats.Study designRandomized, blinded, placebo-controlled crossover study design.AnimalsA total of six purpose-bred, adult female ovariohysterectomized Domestic Short Hair cats.MethodsCats were allocated into three treatments each consisting of two injections, subcutaneous then intravenous (IV) administration, 2 hours apart: treatment SS, two injections of 0.9% saline; treatment BS, buprenorphine (0.24 mg kg^–1, 1.8 mg mL^–1) and saline; and treatment BH, buprenorphine (0.24 mg kg^–1) and hydromorphone (0.1 mg kg^–1). Skin temperature (ST) and thermal threshold (TT) were recorded before (baseline) and for 24 hours following first injection. TT data were analyzed using mixed linear models and a Benjamini–Hochberg sequential adjustment procedure (p < 0.05).ResultsThere were no significant differences among treatments for baseline ST and TT values, treatment SS over time and between treatments BS and BH. Compared with baseline, TT was significantly increased at all time points in treatments BH and BS except at 2 hours in treatment BS. TT was significantly higher than SS at 3–18 hours and 4–12 hours for treatments BS and BH, respectively. Maximal increases in TT were 47.5 °C at 2 hours, 53.9 °C at 3 hours and 52.4 °C at 6 hours in treatments SS, BS and BH, respectively.Conclusions and clinical relevanceAdministration of IV hydromorphone following high-concentration buprenorphine provided no additional antinociception and decreased the duration of effect when compared with high-concentration buprenorphine alone. Alternative analgesics should be considered if additional analgesia is required after administration of high-concentration buprenorphine.     

干扰素诱导跨膜蛋白1对猪源冠状病毒吸附宿主细胞的影响

为探索干扰素诱导跨膜蛋白1（interferon-inducible transmembrane protein 1,IFITM1）对猪源冠状病毒复制的影响,本研究选用猪传染性胃肠炎病毒（Transmissible gastroenteritis virus,TGEV）高致病毒株及其宿主细胞PK15作为研究对象。正常PK15细胞接种TGEV后,实时荧光定量PCR检测感染后不同时间点PK15细胞中一些干扰素刺激基因（interferon-stimulated genes,ISGs）的mRNA表达水平;利用慢病毒表达系统构建稳定表达和稳定干扰IFITM1表达的PK15细胞系。将一段靶向干扰猪源IFITM1的shRNA序列及猪源IFITM1全长,分别插入pLKO.1-EGFP-Puro载体及pLVML-Myc-MCS-IRES-Puro载体中,分别构建出pLKO.1-IFITM1shRNA-EGFP-Puro及pLVML-Myc-IFITM1-IRES-Puro重组质粒。将重组质粒与慢病毒包装质粒共转染293FT细胞后获得带有目的基因的重组慢病毒,慢病毒侵染PK15细胞后用嘌呤霉素进行筛选,获得稳定表达及稳定干扰IFITM1表达的PK15细胞系,分别命名为PK15-IFITM1及PK15-IFITM1^-/-,并分别用实时荧光定量PCR、间接免疫荧光试验（IFA）及Western blotting检测IFITM1干扰效率及表达情况;TGEV接种PK15-IFITM1^-/-和PK15-IFITM1,实时荧光定量PCR测定细胞中TGEV的拷贝数。结果显示,PK15细胞接种TGEV后的48 h内,一些ISGs的mRNA水平均有所上升;PK15-IFITM1^-/-细胞系的干扰效率为70%,PK15-IFITM1细胞系表达成功;在PK15-IFITM1^-/-细胞系中,IFITM1的mRNA水平显著下调,促进了TGEV的复制。反之,在PK15-IFITM1细胞系中,TGEV的复制受到了抑制。但IFITM1的表达或缺失却不影响TGEV对PK15的吸附作用。总之,IFITM1对TGEV有显著的抗病毒作用,IFITM1不影响TGEV对PK15细胞的早期吸附,这为后续IFITM1抗冠状病毒机制的研究奠定了基础。