In vitro and in vivo comparison of five biomaterials used for orthopedic soft tissue augmentation

OBJECTIVE: To compare biomaterials used in orthopedics with respect to in vitro cell viability and cell retention and to in vivo tissue healing and regeneration. ANIMALS: 65 adult female Sprague-Dawley rats and synovium, tendon, meniscus, and bone marrow specimens obtained from 4 adult canine cadavers. PROCEDURES: Synovium, tendon, meniscus, and bone marrow specimens were used to obtain synovial fibroblasts, tendon fibroblasts, meniscal fibrochondrocytes, and bone marrow-derived connective tissue progenitor cells for culture on 5 biomaterials as follows: cross-linked porcine small intestine (CLPSI), non-cross-linked human dermis, cross-linked porcine dermis, non-cross-linked porcine small intestine (NCLPSI), and non-cross-linked fetal bovine dermis. After 1 week of culture, samples were evaluated for cell viability, cell density, and extracellular matrix production. Biomaterials were evaluated in a 1-cm(2) abdominal wall defect in rats. Each biomaterial was subjectively evaluated for handling, suturing, defect fit, and ease of creating the implant at the time of surgery, then grossly and histologically 6 and 12 weeks after surgery. RESULTS: All biomaterials allowed for retention of viable cells in culture; however, CLPSI and NCLPSI were consistently superior in terms of cell viability and cell retention. Cell infiltration for NCLPSI was superior to other biomaterials. The NCLPSI appeared to be replaced with regenerative tissue most rapidly in vivo and scored highest in all subjective evaluations of ease of use. CONCLUSIONS AND CLINICAL RELEVANCE: These data suggested that NCLPSI and CLPSI have favorable properties for further investigation of clinical application in orthopedic tissue engineering.     

Genome-wide identification of microsatellites in white clover (<Emphasis Type="Italic">Trifolium repens</Emphasis> L.) using FIASCO and phpSSRMiner

Background  

Allotetraploid white clover (Trifolium repens L.) is an important forage legume widely cultivated in most temperate regions. Only a small number of microsatellite markers are publicly available and can be utilized in white clover breeding programs. The objectives of this study were to develop an integrated approach for microsatellite development and to evaluate the approach for the development of new SSR markers for white clover.     

Detecting N-myristoylation and S-acylation of host and pathogen proteins in plants using click chemistry

Background

The plant plasma membrane is a key battleground in the war between plants and their pathogens. Plants detect the presence of pathogens at the plasma membrane using sensor proteins, many of which are targeted to this lipophilic locale by way of fatty acid modifications. Pathogens secrete effector proteins into the plant cell to suppress the plant’s defense mechanisms. These effectors are able to access and interfere with the surveillance machinery at the plant plasma membrane by hijacking the host’s fatty acylation apparatus. Despite the important involvement of protein fatty acylation in both plant immunity and pathogen virulence mechanisms, relatively little is known about the role of this modification during plant-pathogen interactions. This dearth in our understanding is due largely to the lack of methods to monitor protein fatty acid modifications in the plant cell.

Results

We describe a rapid method to detect two major forms of fatty acylation, N-myristoylation and S-acylation, of candidate proteins using alkyne fatty acid analogs coupled with click chemistry. We applied our approach to confirm and decisively demonstrate that the archetypal pattern recognition receptor FLS2, the well-characterized pathogen effector AvrPto, and one of the best-studied intracellular resistance proteins, Pto, all undergo plant-mediated fatty acylation. In addition to providing a means to readily determine fatty acylation, particularly myristoylation, of candidate proteins, this method is amenable to a variety of expression systems. We demonstrate this using both Arabidopsis protoplasts and stable transgenic Arabidopsis plants and we leverage Agrobacterium-mediated transient expression in Nicotiana benthamiana leaves as a means for high-throughput evaluation of candidate proteins.

Conclusions

Protein fatty acylation is a targeting tactic employed by both plants and their pathogens. The metabolic labeling approach leveraging alkyne fatty acid analogs and click chemistry described here has the potential to provide mechanistic details of the molecular tactics used at the host plasma membrane in the battle between plants and pathogens.

     

Meat retail conditions within the establishments of Kigali city (Rwanda): bacteriological quality and risk factors for <Emphasis Type="Italic">Salmonella</Emphasis> occurrence

Meat constitutes one of the major vehicles for human foodborne infections. This study aimed to assess the retail conditions and to determine the microbiological quality and safety of meat retailed within the establishments of Kigali (Rwanda). A questionnaire survey was carried out in 150 retail outlets to characterise meat retail conditions. Additionally, 270 retail meat samples were analysed for the enumeration of hygiene indicator bacteria (total mesophilic bacteria and Escherichia coli) and for the qualitative detection of Salmonella, using conventional culture methods. The results revealed that beef was the predominant meat sold within the retail premises of Kigali city, while meat from non-bovine animal species was mainly sold in large establishments. Salmonella was detected in 19.6% of all the retailed meat samples evaluated, whereas the mean loads for total mesophilic bacteria and E. coli were 7.3 and 3.5 log cfu/g, respectively. Three factors, namely the temperature conditions of the meat under retail, the cleanability of the used meat cutting boards, and the training of personnel in hygienic meat handling practices, were found to be significantly (p ≤ 0.05) associated with the risk of Salmonella occurrence in the retailed meat. The findings from this study highlight the need for improvements in hygienic meat handling practices, particularly, in small and medium meat retail establishments in Kigali.     

The prevalence of medial coronoid process disease is high in lame large breed dogs and quantitative radiographic assessments contribute to the diagnosis

Medial coronoid process disease is a common leading cause of thoracic limb lameness in dogs. Computed tomography and arthroscopy are superior to radiography to diagnose medial coronoid process disease, however, radiography remains the most available diagnostic imaging modality in veterinary practice. Objectives of this retrospective observational study were to describe the prevalence of medial coronoid process disease in lame large breed dogs and apply a novel method for quantifying the radiographic changes associated with medial coronoid process and subtrochlear‐ulnar region in Labrador and Golden Retrievers with confirmed medial coronoid process disease. Purebred Labrador and Golden Retrievers (n = 143, 206 elbows) without and with confirmed medial coronoid process disease were included. The prevalence of medial coronoid process disease in lame large breed dogs was calculated. Mediolateral and craniocaudal radiographs of elbows were analyzed to assess the medial coronoid process length and morphology, and subtrochlear‐ulnar width. Mean grayscale value was calculated for radial and subtrochlear‐ulnar zones. The prevalence of medial coronoid process disease was 20.8%. Labrador and Golden Retrievers were the most affected purebred dogs (29.6%). Elbows with confirmed medial coronoid process disease had short (P < 0.0001) and deformed (～95%) medial coronoid process, with associated medial coronoid process osteophytosis (7.5%). Subtrochlear‐ulnar sclerosis was evidenced in ～96% of diseased elbows, with a significant increase (P < 0.0001) in subtrochlear‐ulnar width and standardized grayscale value. Radial grayscale value did not differ between groups. Periarticular osteophytosis was identified in 51.4% of elbows with medial coronoid process disease. Medial coronoid process length and morphology, and subtrochlear‐ulnar width and standardized grayscale value varied significantly in dogs with confirmed medial coronoid process disease compared to controls. Findings indicated that medial coronoid process disease has a high prevalence in lame large breed dogs and that quantitative radiographic assessments can contribute to the diagnosis.     

Atypical actinobacillosis affecting hind limbs and lungs in a single beef cattle herd

Actinobacillosis usually is a sporadic infection that affects the tongue in cattle (“wooden tongue”) with possible spread to the digestive tract. Two 4‐year‐old Rouge‐des‐Prés cows from a single French beef herd were referred for chronic (2‐6 months) swelling and cutaneous nodules in the distal hind limbs. In addition to cutaneous signs, physical examination disclosed cachexia, lameness, lymphadenitis of the hind limbs, and pneumonia in both cows. Cytologic examination of direct skin smears was inconclusive, and no parasites were observed in examination of multiple skin scrapings. Histopathological examination of skin and lung biopsy specimens identified chronic, diffuse, severe pyogranulomatous dermatitis, associated with Splendore‐Hoeppli phenomenon and intralesional Gram‐negative bacteria. Cultures from skin, lymph nodes, and lungs (both cows were euthanized for welfare reasons) identified a Pasteurellaceae organism, confirmed as Actinobacillus lignieresii by partial sequencing of the rpoB gene. This report emphasizes that actinobacillosis can appear as a small outbreak in cattle with cutaneous and respiratory signs.     

An appraisal of the in vivo role of phosphate as a modulator of urinary ammonium and titratable acid excretion in the acidotic rabbit

Previous studies have shown that the intravenous infusion of inorganic phosphate increased urinary ammonium excretion 8‐ to 10‐fold in the acidotic rabbit. This was considered to be a very important observation at the time and to be unique to the rabbit. While investigating this finding, we discovered that the formol titration procedure, used to measure urinary ammonium by this research group, is subject to interference by phosphate, casting doubt on the validity of the urinary ammonium excretion data reported by them in the literature. In order to re‐assess the importance of phosphate as a potential modulator of urinary ammonium excretion in the acidotic rabbit, renal net acid excretion studies were carried out in phosphate‐loaded acidotic animals. We observed that while urinary ammonium excretion increased significantly (p < 0.05) after 50 min of phosphate infusion, the maximum concentrations excreted were substantially less than previously reported in the literature. However, through its urinary buffering capacity, we observed that inorganic phosphate, via an experimentally induced phosphaturia, could substantially enhance titratable acid excretion. Contrary to earlier reports, we demonstrated that phosphate plays a relatively minor in vivo modulator role in enhancing renal net acid excretion through the vehicle of ammonium during acute metabolic acidosis in the hyperphosphataemic rabbit. The findings reported in this study constitute an important update on ammonia metabolism in the acidotic rabbit.