Antagonistic effect of the beneficial bacterium Enterobacter hormaechei against the heavy metal Cu2+ in housefly larvae.

Vermicomposting via housefly larvae can be used to efficiently treat manure and regenerate biofertilizer; however, the uptake of heavy metals could negatively influence the growth and development of larvae. Intestinal bacteria play an important role in the development of houseflies, but their effects on resistance to heavy metal damage in houseflies are still poorly understood. In this study, the life history traits and gut microbiota of housefly larvae were evaluated after exposure to an environment with Cu2+ -Enterobacter hormaechei. The data showed that exposure to 300 µg/mL Cu2+ significantly inhibited larval development and locomotor activity and reduced immune capacity. However, dietary supplementation with a Cu2+ -Enterobacter hormaechei mixture resulted in increased body weight and length, and the immune capacity of the larvae returned to normal levels. The abundances of Providencia and Klebsiella increased when larvae were fed Cu2+ -contaminated diets, while the abundances of Enterobacter and Bacillus increased when larvae were exposed to a Cu2+ -Enterobacter hormaechei mixture-contaminated environment. In vitro scanning electron microscopy analysis revealed that Enterobacter hormaechei exhibited obvious adsorption of Cu2+ when cultured in the presence of Cu2+, which reduced the damage caused by Cu2+ to other bacteria in the intestine and protected the larvae from Cu2+ injury. Overall, our results showed that Enterobacter hormaechei can absorb Cu2+ and increase the abundance of beneficial bacteria, thus protecting housefly larvae from damage caused by Cu2+. These results may fill the gaps in our understanding of the interactions between heavy metals and beneficial intestinal bacteria, offering valuable insights into the interplay between housefly larvae and metal contaminants in the environment. This approach could enhance the efficiency of converting manure contaminated with heavy metals to resources using houseflies.

Characterization of the fatty acid binding protein 3 (FABP3) promoter and its transcriptional regulation by cAMP response element binding protein 1 (CREB1) in goat mammary epithelial cells.

Fatty acid binding protein 3 (FABP3) is involved in signal transduction pathways, and in the uptake and utilization of long-chain fatty acids. However, the transcriptional regulation of FABP3 in goat is unclear. In this study, the FABP3 5' flanking region was amplified from goat (Capra hircus) genomic DNA. Luciferase reporter vectors containing promoter fragments of five different lengths were constructed and transfected into dairy goat mammary epithelial cells. The region of the promoter located between -1801 and -166 bp upstream of the transcription start site (TSS) exhibited the highest luciferase activity, and contained two cAMP response elements (CREs) located at -1632 bp and -189 bp. Interference with CREB1 significantly downregulated FABP3 promoter activity. In addition, FABP3 promoter activity was significantly reduced after mutation of the CRE1 (-1632 bp) and CRE2 (-189 bp) sites. Further analysis indicated that the CRE2 site was essential for the transcriptional activity induced by CREB1. These results demonstrated that CREB1 is involved in the transcriptional regulation of FABP3 expression in the goat mammary gland via a direct mechanism, thus revealing a novel signaling pathway involved in fatty acid metabolism in goat.

Molecular epidemiology of type I and II feline coronavirus from cats with suspected feline infectious peritonitis in China between 2019 and 2021.

Feline infectious peritonitis (FIP) is one of the deadliest diseases of cats in China. In this study, 120 ascitic fluid samples from FIP-suspected cats were collected from veterinary hospitals in 21 provinces in China between 2019 and 2021. One hundred nine samples were positive for feline coronavirus (FCoV), with no feline immunodeficiency virus infections and one feline leukemia virus infection (1/109, 0.92%). The prevalence of FCoV was significantly associated with age (p < 0.01) and was not highly associated with gender, breed, geographical location, or viral coinfection (p > 0.01). One unique strain, SD/202012/003, contained a six-nucleotide deletion in the spike gene. Sequence analysis showed that 94.68% (89/94) of the isolates had a mutation of methionine to leucine at position 1058 in the spike protein. The epidemiological data obtained of FCoV in this study may be beneficial for clinical monitoring of FCoV in China.

Acyl-CoA synthetase short-chain family member 2 (ACSS2) is regulated by SREBP-1 and plays a role in fatty acid synthesis in caprine mammary epithelial cells.

Sterol regulatory element binding protein 1 (SREBP-1) is well-known as the master regulator of lipogenesis in rodents. Acyl-CoA synthetase short-chain family member 2 (ACSS2) plays a key role in lipogenesis by synthesizing acetyl-CoA from acetate for lipogenesis. ATP citrate lyase (ACLY) catalyzes the conversion of citrate and coenzyme A to acetyl-CoA, hence, it is also important for lipogenesis. Although ACSS2 function in cancer cells has been elucidated, its essentiality in ruminant mammary lipogenesis is unknown. Furthermore, ACSS2 gene promoter and its regulatory mechanisms have not known. Expression of ACSS2 was high in lipid synthesizing tissues, and its expression increased during lactation compared with non-lactating period. Simultaneous knockdown of both ACSS2 and ACLY by siRNA in primary goat mammary epithelial cells decreased (p < 0.05) the mRNA abundance of genes associated with de novo fatty acid synthesis (FASN, ACACA, SCD1) and triacylglycerol (TAG) synthesis (DGAT1, DGAT2, GPAM, and AGPAT6). Genes responsible for lipid droplet formation and secretion (PLIN2 and PLIN3) and fatty acid oxidation (ATGL, HSL, ACOX, and CPT1A) all decreased (p < 0.05) after ACSS2 and ACLY knockdown. Total cellular TAG content and lipid droplet formation also decreased. Use of a luciferase reporter assay revealed a direct regulation of ACSS2 by SREBP-1. Furthermore, SREBP-1 interacted with an SRE (SREBP response element) spanning at -475 to -483 bp on the ACSS2 promoter. Taken together, our results revealed a novel pathway that SREBP-1 may regulate fatty acid and TAG synthesis by regulating the expression of ACSS2.

MiR-145 Regulates Lipogenesis in Goat Mammary Cells Via Targeting INSIG1 and Epigenetic Regulation of Lipid-Related Genes.

MicroRNAs (miRNAs) are noncoding RNA molecules that regulate gene expression at the post-transcriptional level to cause translational repression or degradation of targets. The profiles of miRNAs across stages of lactation in small ruminant species such as dairy goats is unknown. A small RNA library was constructed using tissue samples from mammary gland of Saanen dairy goats harvested at mid-lactation followed by sequencing via Solexa technology. A total of 796 conserved miRNAs, 263 new miRNAs, and 821 pre-miRNAs were uncovered. After comparative analyses of our sequence data with published mammary gland transcriptome data across different stages of lactation, a total of 37 miRNAs (including miR-145) had significant differences in expression over the lactation cycle. Further studies revealed that miR-145 regulates metabolism of fatty acids in goat mammary gland epithelial cells (GMEC). Compared with nonlactating mammary tissue, lactating mammary gland had a marked increase in expression of miR-145. Overexpression of miR-145 increased transcription of genes associated with milk fat synthesis resulting in greater fat droplet formation, triacylglycerol accumulation, and proportion of unsaturated fatty acids. In contrast, silencing of miR-145 impaired fatty acid synthesis. Inhibition of miR-145 increased methylation levels of fatty acid synthase (FASN), stearoyl-CoA desaturase 1 (SCD1), peroxisome proliferator-activated receptor gamma (PPARG), and sterol regulatory element binding transcription factor 1 (SREBF1). Luciferase reporter assays confirmed that insulin induced gene 1 (INSIG1) is a direct target of miR-145. These findings underscore the need for further studies to evaluate the potential for targeting miR-145 for improving beneficial milk components in ruminant milk. J. Cell. Physiol. 232: 1030-1040, 2017. © 2016 Wiley Periodicals, Inc.

SCD1 Alters Long-Chain Fatty Acid (LCFA) Composition and Its Expression Is Directly Regulated by SREBP-1 and PPARγ 1 in Dairy Goat Mammary Cells.

Stearoyl-CoA desaturase 1 (SCD1) is a key enzyme for the synthesis of the monounsaturated fatty acids (MUFA) palmitoleic acid and oleic acid. In non-ruminant species, SCD1 expression is known to be tightly regulated by a variety of transcription factors. Although the role of SCD1 and the transcriptional regulatory mechanism by SREBP-1 and PPARs in other species is clear, changes in lipid metabolism related to SCD1 and via the regulation of SREBP-1 or PPARG1 in ruminant mammary tissue remain largely unknown. Here, we demonstrated that SCD1 expression in goat mammary tissue is higher during lactation than the dry period. Overexpression of SCD1 increased the intracellular MUFA content and lipid accumulation, whereas SCD1 silencing resulted in a significant decrease in oleic acid concentration and triacylglycerol (TAG) accumulation. The overexpression of SREBF1 in goat mammary epithelial cells (GMEC) enhanced SCD1 expression and its promoter activity, but that effect was abolished when SREBF1 was silenced. Furthermore, deletion of sterol regulatory element (SRE) and the nuclear factor (NF-Y)-binding sites within a -1713 to +65-base pair region of the SCD1 promoter completely abolished SREBP-1-induced SCD1 transcription. Otherwise, PPARG1 overexpression also stimulated the expression of SCD1 and its transcriptional activity directly via a PPAR response element (PPRE) in the SCD1 promoter. Together, these results indicate that SCD1 could markedly affect the fatty acid composition and rate of TAG synthesis through direct regulation via SREBP-1 and PPARG1, hence, underscoring an important role of the enzyme and this transcription regulator in controlling mammary gland lipid synthesis in the goat. J. Cell. Physiol. 232: 635-649, 2017. © 2016 Wiley Periodicals, Inc.

miR-26b promoter analysis reveals regulatory mechanisms by lipid-related transcription factors in goat mammary epithelial cells.

MicroRNA (miRNA) regulate protein abundance and control diverse aspects of cellular processes and biological functions associated with lipid metabolism. MiR-26b and its host gene CTDSP1 regulate triacylglycerol synthesis by synergistically suppressing the insulin-induced gene 1 (INSIG1); however, the direct regulators of miR-26b expression remain unknown. In the present study, we characterized the activity of a novel putative promoter region in miR-26b. Results revealed that promoter activity and miR-26b expression are dynamically regulated by different transcription factors including peroxisome proliferator-activated receptor gamma (PPARG), sterol regulatory element binding transcription factor 1 (SREBF1), and liver X receptor α (LXRα). Two binding sites for the SREBF1 (SRE1 and SRE3) and the PPARG (peroxisome proliferator response element 1 and 2; PPRE1 and PPRE2), respectively, were identified in the miR-26b promoter, which demonstrated that those binding sites are responsible for the activation by PPARG and SREBF1. In silico analysis and site-directed mutagenesis of LXRα binding elements (LXRE) and SREBF1 binding elements (SRE) revealed that the effects of Ad-LXRα + T0901317 requires the presence of SRE, whereas potential LXRE had no effects on miR-26b expression. This suggested that regulation of miR-26b by LXRα is indirectly via an SRE, and miR-26b is regulated by transcription factors dually through DNA methylation and directly through binding to its promoter, all of which implies that regulation of miR-26b in ruminant mammary epithelial cells results from various mechanisms. In conclusion, we demonstrate a novel dual-regulatory mechanism whereby transcription factors regulate the expression of miR-26b. Overall, these findings contribute to our understanding of the interactions between specific promoter elements and the control of transcription and translation of miRNA.

MicroRNA-26a/b and their host genes synergistically regulate triacylglycerol synthesis by targeting the INSIG1 gene.

The microRNA-26 (miR-26) family is known to control adipogenesis in non-ruminants. The genomic loci of miR-26a and miR-26b have been localized in the introns of genes encoding for the proteins of the C-terminal domain RNA polymerase II polypeptide A small phosphatase (CTDSP) family. Insulin-induced gene 1 (INSIG1) encodes a protein with a key role in the regulation of lipogenesis in rodent liver. In the present study, we investigated the synergistic function of the miR-26 family and their host genes in goat mammary epithelial cells (GMEC). Downregulation of miR-26a/b and their host genes in GMEC decreased the expression of genes relate to fatty acid synthesis (PPARG, LXRA, SREBF1, FASN, ACACA, GPAM, LPIN1, DGAT1 and SCD1), triacylglycerol accumulation and unsaturated fatty acid synthesis. Luciferase reporter assays confirmed INSIG1 as a direct target of miR-26a/b. Furthermore, inhibition of the CTDSP family also downregulated the expression of INSIG1. Taken together, our findings highlight a functional association of miR-26a/b, their host genes and INSIG1, and provide new insights into the regulatory network controlling milk fat synthesis in GMEC. The data indicate that targeting this network via nutrition might be important for regulating milk fat synthesis in ruminants.

Genes regulating lipid and protein metabolism are highly expressed in mammary gland of lactating dairy goats.

Dairy goats serve as an important source of milk and also fulfill agricultural and economic roles in developing countries. Understanding the genetic background of goat mammary gland is important for research on the regulatory mechanisms controlling tissue function and the synthesis of milk components. We collected tissue at four different stages of goat mammary gland development and generated approximately 25 GB of data from Illumina de novo RNA sequencing. The combined reads were assembled into 51,361 unigenes, and approximately 60.07 % of the unigenes had homology to other proteins in the NCBI non-redundant protein database (NR). Functional classification through eukaryotic Ortholog Groups of Protein (KOG), gene ontology (GO), and Kyoto Encyclopedia of Genes and Genomes (KEGG) revealed that the unigenes from goat mammary glands are involved in a wide range of biological processes and metabolic pathways, including lipid metabolism and lactose metabolism. The results of qPCR revealed that genes encoding FABP3, FASN, SCD, PLIN2, whey proteins (LALBA and BLG), and caseins (CSN1S1, CSN1S2, CSN2 and CSN3) at 100 and 310 days postpartum increased significantly compared with the non-lactating period. In addition to their role in lipid and protein synthesis, the higher expression at 310 days postpartum could contribute to mammary cell turnover during pregnancy. In conclusion, this is the first study to characterize the complete transcriptome of goat mammary glands and constitutes a comprehensive genomic resource available for further studies of ruminant lactation.

Establishment and Characterization of a New Cell Line from Enzootic Nasal Adenocarcinoma in Goats: ENA-1.

Enzootic nasal adenocarcinoma (ENA) is a contagious tumor disease of goats and sheep, which is caused by enzootic nasal tumor virus (ENTV). To better understand the pathogenesis of ENA, this study aimed to establish a goat ENA cell line (ENA-1). The cells have been characterized with regard to morphology, growth rate, ultrastructural features, chromosome number, expression of CK7 and CK18, tumorigenicity, species, and mycoplasma contamination. ENA-1 had an epithelioid cell morphology with an unstable chromosome number under a light microscope. Under an electron microscope, the cell nuclear heterogeneity was not obvious, and there were more intermediate filaments and a small number of immature retrovirus-like particles in the cytoplasm. ENA-1 had strong proliferative potential, and the cell multiplication time was about 36 h, which could make BALB/c nude mice develop tumors. CK7 and CK18 were expressed in the cytoplasm of primary goat tumors, in transplanted tumors from nude mice, and un ENA-1 cells with the same intensity. PCR revealed that ENA-1 continuously carried ENTV-2 up to the 17th generation with no germline contamination or mycoplasma contamination. In conclusion, using a serum-containing culture system, ENA-1 cells were successfully isolated, cultured, and purified from goat tumor tissues. The isolated ENA-1 cells retained robust proliferation potential and maintained their phenotype, indicating the potential application of the ENA-1 cell line as an in vitro model of ENA.

Application of bacteria and bacteriophage cocktails for biological control of houseflies.

BACKGROUND: Houseflies, Musca domestica L., are an ubiquitous pest that can transmit numerous diseases and threaten human health. Increasing insecticide resistance shown by houseflies necessitates the develop new control alternatives. The housefly gut is densely colonized with microorganisms that interact with each other dynamically and benefit the host's health. However, the impact of multiple symbiotic bacteria on the composition of housefly gut microbiota and the host's activities remains unclear. METHODS: We isolated and cultured 12 bacterial species from the intestines of housefly larvae. We also isolated seven bacteriophages to precisely target the regulation of certain bacterial species. Using 16S rRNA high-throughput gene sequencing, we analyzed the bacterial diversity after orally administering bacteria/phage cocktails to houseflies. RESULTS: Our results showed that larval growth was promoted, the abundance of beneficial bacteria, such as Klebsiella and Enterobacter, was increased and the abundance of harmful bacteria, such as Providencia, Morganella and Pseudomonas, was decreased in housefly larvae fed with the beneficial bacteria cocktail. However, oral administration of both beneficial and harmful bacterial phage cocktails inhibited larval growth, probably due to the drastic alteration of gut flora. Untargeted metabolomics using liquid chromatography-mass spectrometry showed that disturbances in gut microbiota changed the larval metabolite profiles. Feeding experiments revealed that disrupting the intestinal flora suppressed the beneficial bacteria and increased the harmful bacteria, causing changes in the metabolites and inhibiting larval growth. CONCLUSIONS: Based on our results, bacteria/phage cocktails are effective tools for regulating the intestinal flora of insects and have a high potential as a biological control agent for incorporation into an integrated pest management program.

Insights into Transcriptomic Differences in Ovaries between Lambs and Adult Sheep after Superovulation Treatment.

Superovulation technology shows a great potential for shortening breeding time. Using the juvenile superovulation technology, juvenile animals can generate more follicles than adult animals. By sequencing using high-throughput methods, we studied and described differentially expressed (DE) long non-coding RNA (lncRNAs) and messenger RNAs (mRNAs) in the ovaries of young and adult sheep. Herein, 242 DE lncRNAs and 3150 DE mRNAs were screened. Through GO and KEGG analyses, we obtained genes related to ovarian/follicle development and ovulation in DE mRNAs, including OaFSHR, OaLHCGR, OaLDLR, OaZP3, OaSCARB1, and OaPDGFRA; through lncRNA-mRNA correlation analysis, we found that genes associated with ovarian/follicle development or ovulation include: XR_003585520.1, MSTRG.15652.1, XR_003588840.1, and their paired genes PDGFC, LRP5, and LRP1. We observed a synergistic effect between PDGFR and LRP1. PDGFR may play a leading role compared with LRP1. The induced LHCGR in lambs is higher than in adult sheep, showing more sensitivity to LH. The release of the oocytes was stimulated. Among the three lncRNAs, we found that XR_003588840.1 was significantly different and might perform a regulatory role in ovarian/follicle growth or ovulation.

What do users and their aiding professionals want from future devices in upper limb prosthetics? A focus group study.

BACKGROUND: High rejection rates of upper limb prosthetics indicate that current prosthetic devices only partially meet user demands. This study therefore investigated the benefits and challenges with current prostheses, associated services and potential areas for improvement from the perspective of upper limb prosthesis users and various professionals working in the field of upper limb and hand prosthetics. METHODS AND FINDINGS: Seven different focus group discussions were conducted with 32 participants. Participants were grouped by prosthesis type, if they were prosthesis users, or professionals. All focus group discussions were transcribed verbatim, and a summarizing content analysis was performed. Three main topic areas to be addressed emerged from the interviews: 1. a properly functioning prosthesis, 2. the infrastructure, and 3. users' psychological and physical prerequisites. The interaction between a well-functioning prosthesis and a well-developed infrastructure was shown to be important for successful use. CONCLUSIONS: Our study raises many of the same issues that have been reported in previous qualitative studies, dating back over several decades. This study underlines the need to include users and professionals in the future development of prosthetic devices.

Scalable synthesis of Cu clusters for remarkable selectivity control of intermediates in consecutive hydrogenation.

Subnanometric Cu clusters that contain only a small number of atoms exhibit unique and, often, unexpected catalytic behaviors compared with Cu nanoparticles and single atoms. However, due to the high mobility of Cu species, scalable synthesis of stable Cu clusters is still a major challenge. Herein, we report a facile and practical approach for scalable synthesis of stable supported Cu cluster catalysts. This method involves the atomic diffusion of Cu from the supported Cu nanoparticles to CeO2 at a low temperature of 200 °C to form stable Cu clusters with tailored sizes. Strikingly, these Cu clusters exhibit high yield of intermediate product (95%) in consecutive hydrogenation reactions due to their balanced adsorption of the intermediate product and dissociation of H2. The scalable synthesis strategy reported here makes the stable Cu cluster catalysts one step closer to practical semi-hydrogenation applications.

Aerobic and facultative anaerobic Klebsiella pneumoniae strains establish mutual competition and jointly promote Musca domestica development.

Introduction: The gut microenvironment in housefly harbors a rich and diverse microbial community which plays a crucial role in larval development. However, little is known about the impact of specific symbiotic bacteria on larval development as well as the composition of the indigenous gut microbiota of housefly. Methods: In the present study, two novel strains were isolated from housefly larval gut, i.e., Klebsiella pneumoniae KX (aerobe) and K. pneumoniae KY (facultative anaerobe). Moreover, the bacteriophages KXP/KYP specific for strains KX and KY were used to analyse the effects of K. pneumoniae on larval development. Results: Our results showed that dietary supplementation with K. pneumoniae KX and KY individually promoted housefly larval growth. However, no significant synergistic effect was observed when the two bacterial strains were administered in combination. In addition, using high-throughput sequencing, it was demonstrated that the abundance of Klebsiella increased whereas that of Provincia, Serratia and Morganella decreased when housefly larvae received supplementation with K. pneumoniae KX, KY or the KX-KY mixture. Moreover, when used combined, K. pneumoniae KX/KY inhibited the growth of Pseudomonas and Providencia. When the abundance of both bacterial strains simultaneously increased, a balance in total bacterial abundance was reached. Discussion: Thus, it can be assumed that strains K. pneumoniae KX and KY maintain an equilibrium to facilitate their development in housefly gut, by establishing competition but also cooperation with each other to maintain the constant composition of gut bacteria in housefly larvae. Thus, our findings highlight the essential role of K. pneumoniae in regulating the composition of the gut microbiota in insects.

Microsatellite instability in Marek's disease virus infected primary chicken embryo fibroblasts.

BACKGROUND: Marek's disease virus (MDV), an oncogenic α-herpes virus, causes a devastating disease in chickens characterized by development of lymphoblastoid tumors in multiple organs. Microsatellite instability (MSI), a symptom of defect in DNA mismatch repair function, is a form of genomic instability frequently detected in many types of tumors. However, the involvement of MSI in MDV-infected cells has not been investigated. In this study, we determined the presence and frequency of MSI in primary chicken embryo fibroblasts infected with or without MDV strain in vitro. RESULTS: 118 distinct microsatellite markers were analyzed by polymerase chain reaction (PCR) in 21 samples. MSI was found in 91 of 118 markers, and 12 out of 118 demonstrated frequency of MSI at ≥ 40%. 27 of 118 microsatellite loci did not show microsatellite instability. CONCLUSIONS: These findings showed that MSI was a real event occurring in primary chicken embryo fibroblasts infected with MDV in vitro as evidenced by the high frequency of MSI, and may be specifically associated with genome alteration of host cells during MDV infected.

Production and characterization of alkaline protease from hemoglobin-degrading Bacillus pumilus NJM4 to produce fermented blood meal.

The aim of the research was to isolate the hemoglobin-degrading bacterial strain to produce fermented blood meal and to characterize the protease produced by this strain. The strain NJM4, a kind of hemoglobin-degrading bacterial strain, was isolated by blood agar plates from slaughterhouse and identified as a Bacillus pumilus by physiological, biochemical, and morphological characteristics and by 16S rRNA gene sequencing. Bacillus pumilus NJM4 could degrade hemoglobin up to 85% in 36 h under the laboratory conditions. The optimal conditions for protease production was achieved at an initial pH level of 8.67, inoculum size of 4%, incubation temperature of 37 °C, and agitation rate 200 rpm. The optimum pH and temperature of hemoglobin-degrading proteases were at 9.0 and 50 °C, respectively. The protease activity was slightly decreased in presence of Ca(2+) and DTT. It was significantly inhibited in the presence of PMSF and EDTA identifying it as alkaline serine-metalloproteinase. Bacillus pumilus NJM4 and hemoglobin-degrading proteases provide potential use for biotechnological process of fermentation and enzymolysis blood meal as animal feed supplement.

Knee disarticulation following oncologic total knee arthroplasty: A 5-year follow-up case report.

BACKGROUND: The present case report describes the 5-year follow-up results of an atypical knee disarticulation of a man previously treated with an oncologic total knee arthroplasty due to an Ewing sarcoma. METHODS: The patient presented an aseptic loosened tibial component of a tumor prosthesis system and requested final amputation, as he had previously suffered from five revision surgeries. To encourage the most functional outcome regarding an exoskeletal prosthesis, we decided to disarticulate the knee joint while retaining the currently fixed femoral component to create a full end-bearing stump. FINDINGS: The patient could be mobilized as a functional knee disarticulated amputee. Seven months after amputation, he showed a slightly less symmetrical gait compared to the preoperative status (preoperative mean Symmetry Index: 0.984 for kinematics and 0.940 for kinetics, 7-month postoperative Symmetry Index: 0.858 and 0.915). At the 5-year follow-up, the femoral component is still stably fixated and shows no loosening signs. In addition, the Symmetry Index increased to 0.908 and 0.949. INTERPRETATION: Even after 5 years, the presented amputation appears to be consistent with "conventional" knee disarticulation. The femoral component still withstands the altered loads and the patient shows a further improved gait pattern.

EVALUATION OF THE INHIBITORY EFFECTS OF SIX NATURAL PRODUCT EXTRACTS AGAINST BABESIA GIBSONI IN VITRO AND IN VIVO.

New antibabesial drugs are required to fight resistant parasites, and plant-derived natural products are a robust source. Six kinds of natural product extracts derived from herbal medicines that are traditionally used for the treatment of malaria were selected to test the antibabesial effect on Babesia gibsoni in vitro and in vivo. Parasitized blood was collected from dogs infected with B. gibsoni to evaluate the inhibitory effect of verbenalin, catechin hydrate, dihydrolycorine, embelin, ursolic acid, agrimol B, and bruceine H in vitro. The expression levels of the 18S rRNA gene in all drug-treated groups were determined by relative quantification using a real-time PCR method. Significant inhibition of the in vitro growth of B. gibsoni was observed after treatment by those natural product extracts (200 nM concentration) (P < 0.05). Catechin hydrate showed the highest activity in vitro due to the lowest expression levels of the 18S rRNA gene. The IC50 value of catechin hydrate against B. gibsoni was 273 nM. In B. gibsoni infected dogs, intravenous administrations of catechin hydrate and diminazene aceturate showed significant (P < 0.05) inhibition of B. gibsoni growth at a dose of 11 mg/kg and 10 mg/kg, respectively, compared to the control group. The results of our study may suggest that catechin hydrate may be a promising alternative to treat canine babesiosis caused by B. gibsoni.

Radiography, CT, and MRI Diagnosis of Enzootic Nasal Tumor in Goats Infected With Enzootic Nasal Tumor Virus.

The aim of this study was to describe radiography, computed tomography (CT), and magnetic resonance imaging (MRI) findings of enzootic nasal tumors in goats infected with enzootic nasal tumor viruses. Five of six goats with a mean age of 2 years, showed clinical signs of respiratory disease. Head radiographs showed increased density of the unilateral or bilateral nasal cavity in four goats, and a CT scan showed that the space-occupying lesion of the nasal cavity originated from the ethmoid bone and was enhanced homogeneously postcontrast in all goats. The nasal concha was destroyed and the paranasal sinus mucosa was thickened and filled with fluid in some goats. On MRI, the mass exhibited equal or slightly higher signal intensity on T2 weighted images, equal signal intensity on T1 weighted images, a high signal on fluid-attenuated inversion recovery images and heterogeneous enhancement postcontrast. After dissection, histopathological examination of the mass and virus genome detection of the nasal secretions confirmed that the intranasal mass was a low-grade adenocarcinoma and that the goats were infected with enzootic nasal tumor virus type 2. In conclusion, CT and MRI have high diagnostic values for enzootic nasal tumors because they match the postmortem findings and are more accurate than radiography.