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Biomedical subjects

D Marshall

Publications and source records attributed to D Marshall.

At least 19 recordsLinked to original sources
PubMed2006

Pm34: a new powdery mildew resistance gene transferred from Aegilops tauschii Coss. to common wheat (Triticum aestivum L.).

Powdery mildew is a major fungal disease in wheat growing areas worldwide. A novel source of resistance to wheat powdery mildew present in the germplasm line NC97BGTD7 was genetically characterized as a monogenic trait in greenhouse and field trials using F(2) derived lines from a NC97BGTD7 X Saluda cross. Microsatellite markers were used to map and tag this resistance gene, now designated Pm34. Three co-dominant microsatellite markers linked to Pm34 were identified and their most likely order was established as: Xbarc177-5D, 5.4cM, Pm34, 2.6cM, Xbarc144-5D, 14cM, Xgwm272-5D. These microsatellite markers were previously mapped to the long arm of the 5D chromosome and their positions were confirmed using Chinese Spring nullitetrasomic Nulli5D-tetra5A and ditelosomic Dt5DL lines. Pm2, the only other known Pm gene on chromosome 5D, has been mapped to the short arm and its specificity is different from that of Pm34.

Ascomycota↗
PubMed2006

Clinicopathologic features of early adenocarcinoma of the cervix initially managed with cervical conization.

OBJECTIVE: To evaluate the clinicopathologic features of microinvasive adenocarcinoma of the cervix in order to guide the management of patients with this disease. MATERIALS AND METHODS: A retrospective review was conducted of patients diagnosed with early invasive, 1 mm and 2 mm and 3 mm and 4 mm and <or=5 mm. Three patients were treated with a conization only, 4 patients were treated with a simple hysterectomy, 25 patients were treated with a radical hysterectomy (RH) and PLN dissection (PLND), and 1 patient was treated with a radical trachelectomy and PLND. Ten patients had positive conization margins for invasive cancer, 3 patients had margins positive for adenocarcinoma in situ, 14 patients had negative margins, and in 6 patients the margin status could not be evaluated. Of the 10 patients with positive margins, 5 of 10 (50%) had residual disease in the subsequent surgical specimen. Three patients who underwent definitive management with conization alone originally had positive margins, underwent a second repeat conization, and are included in this group. Of the 16 patients with negative margins, no patient had residual disease in a subsequent surgical specimen. Of the 25 patients who underwent a RH and PLND, none had parametrial involvement and none had PLN involvement. All patients remained without evidence of disease at median follow-up of 30 months. CONCLUSIONS: Historically, the standard management of early invasive adenocarcinoma of the cervix has been controversial, and some clinicians continue to favor radical treatments. Based on the absence of parametrial spread and PLN involvement in early lesions, physicians and patients should consider treatment with conization with negative margins (when future fertility is desired) or simple hysterectomy. Prospective studies are required to document the safety of this approach.

Adenocarcinoma↗
PubMed2005

A double-blind randomized controlled trial comparing alternate forms of high molecular weight hyaluronan for the treatment of osteoarthritis of the knee.

OBJECTIVE: To compare the safety and effectiveness of a high molecular weight hyaluronan produced by biological fermentation (Bio-HA) with those of avian-derived hyaluronan that uses cross-linking to achieve high molecular weight (CL-HA). DESIGN: This was a prospective, multicenter, randomized, double-blind trial evaluating patients with confirmed osteoarthritis of the knee. The Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC Index) pain subscale was the primary effectiveness measure (visual analog scale). Both products were administered via three weekly injections, with follow-up evaluations at weeks 3, 6 and 12. Acetaminophen was permitted as rescue medication and quantitated by pill counts. RESULTS: Analyses were performed on the intent-to-treat population, defined as all patients receiving at least one injection. Of the 321 patients randomized to treatment, 314 patients (98%) completed the final study assessment. Improvement in the average WOMAC Index pain score was 29.8mm (-61.6%) for Bio-HA and 28.8mm (-54.9%) for CL-HA, meeting the prospective criteria for non-inferiority. For the secondary outcome measures, statistically significant differences favored Bio-HA for the number of patients requiring acetaminophen (P=0.013) and patient global satisfaction evaluations (P=0.03). Local reactions differed between the products in that 15 effusions were reported in 13 CL-HA patients (8.1%) after injection, compared to one effusion (0.6%) after Bio-HA injection (P=0.0015). CONCLUSION: The effectiveness of Bio-HA was not inferior to that of CL-HA. The significantly higher incidence of post-injection effusion in the CL-HA group provides a safety advantage for Bio-HA. These data suggest that Bio-HA has an improved benefit-risk profile compared with CL-HA.

Adjuvants, Immunologic↗
PubMed2005

Examination of the molecular signature associated with islet dysfunction.

The aim of this study was to identify the molecular signatures that are predictive of nonfunctional islet preparations. We examined functional outcomes of six islet preparations accepted for research purposes from human donors. Islet were maintained on culture in M-SFM media for 7 to 14 days then transplanted into NOD-SCID mice. At the time of transplant, RNA was extracted from a second aliquot of cultured islets for expression analysis. We also performed gene expression analysis using high-density Affymetrix U133A GeneChips on these preparations. Among 1833 genes selected, hierarchical clustering was performed using the GeneSpring software package (Silicon Genetics, Inc.), where 754 genes (higher in nonfunctional) and 177 genes (lower in nonfunctional) were differentially expressed with tight pattern of expression. Islets with low functionality showed high relative levels of expression of hypoxia-induced genes and increased frequency of expression of proinflammatory and proangiogenic genes, such as vascular endothelial growth factor. Conversely, nonfunctional islets had low levels of insulin-processing message. The general profile of these low-functionality islets shows attempted recovery from hypoxic assault and little effort directed toward insulin production and secretion. Further identification of the molecular signature of nonfunctional islets could allow the development of a potency assay for human transplantation.

Cadaver↗
PubMed2005

Using a 360 degrees diagnostic screening tool to provide an evidence trail of junior doctor performance throughout their first postgraduate year.

New medical graduates in the UK are known as Preregistration House Officers (PRHOs). At the end of this first postgraduate year, the Postgraduate Dean is responsible for allowing the PRHO to be fully registered with the General Medical Council. During the period 1999-2002 Professor Miriam Friedman Ben-David designed a more robust approach to appraisal and assessment of PRHOs, which provided educational feedback to all trainees, allowed any poor performers to be detected at an early stage, and provided 'hard' observable evidence for certification decisions. This paper describes the more recent development of her work resulting from further piloting of her system. The key tool, a 360 degrees diagnostic questionnaire, is designed to identify strengths and weaknesses in individual performance. It is presently being used as a screening tool to identify any trainees needing additional support and further assessment. The tool also forms part of an evidence trail for all PRHOs and helps inform formative assessment as well as contributing, along with other evidence, to full registration decisions. The evaluation of this tool, also described in this paper, shows the feasibility of implementing such a system on a wider scale and illustrates the successful balance made between robustness and feasibility.

Attitude of Health Personnel↗
PubMed2004

Latent S alleles are widespread in cultivated self-compatible Brassica napus.

The genetic control of self-incompatibility in Brassica napus was investigated using crosses between resynthesized lines of B. napus and cultivars of oilseed rape. These crosses introduced eight C-genome S alleles from Brassica oleracea (S16, S22, S23, S25, S29, S35, S60, and S63) and one A-genome S allele from Brassica rapa (SRM29) into winter oilseed rape. The inheritance of S alleles was monitored using genetic markers and S phenotypes were determined in the F1, F2, first backcross (B1), and testcross (T1) generations. Two different F1 hybrids were used to develop populations of doubled haploid lines that were subjected to genetic mapping and scored for S phenotype. These investigations identified a latent S allele in at least two oilseed rape cultivars and indicated that the S phenotype of these latent alleles was masked by a suppressor system common to oilseed rape. These latent S alleles may be widespread in oilseed rape varieties and are possibly associated with the highly conserved C-genome S locus of these crop types. Segregation for S phenotype in subpopulations uniform for S genotype suggests the existence of suppressor loci that influenced the expression of the S phenotype. These suppressor loci were not linked to the S loci and possessed suppressing alleles in oilseed rape and non-suppressing alleles in the diploid parents of resynthesized B. napus lines.

Alleles↗
PubMed2003

MRL/lpr lupus-prone mice show exaggerated ICAM-1-dependent leucocyte adhesion and transendothelial migration in response to TNF-alpha.

OBJECTIVE: Endothelial activation and dysfunctional leucocyte-endothelial interactions are thought to play key roles in the pathogenesis of systemic lupus erythematosus (SLE). The object of this study was to investigate directly the effect of increased endothelial adhesion molecule expression on leucocyte-endothelial cell interactions, using the MRL/lpr mouse model. METHODS: Leucocyte rolling, arrest and transendothelial migration were quantified in the cremaster muscle microcirculation of 20-week-old MRL/lpr mice, using intravital microscopy. Endothelial adhesion molecule expression was quantified using intravenously injected radiolabelled monoclonal antibodies. RESULTS: Basal expression of intercellular adhesion molecule 1 (ICAM-1) by cremaster endothelium was 2-fold greater in MRL/lpr than in MRL/++ mice (P<0.05). There was a 1.6-fold increase in expression of vascular adhesion molecule 1 (VCAM-1), but no increase in E-selectin or P-selectin expression. Following intrascrotal injection of saline, no difference was detected in leucocyte-endothelial interactions between MRL/lpr and control MRL/++ mice. In contrast, intrascrotal injection of tumour necrosis factor alpha (TNF-alpha) (2 h test period) led to significantly increased numbers of adherent and extravasated leucocytes in MRL/lpr (5.98+/-0.71 and 5.45+/-0.34 leucocytes per 100 micro m vessel segment respectively) compared with MRL/++ mice (3.63+/-0.26 and 2.97+/-0.24 respectively, each P<0.05). Treatment of TNF-alpha-stimulated mice with anti-ICAM-1 F(ab')2 (YN1) abolished the difference between MRL/lpr and MRL/++ mice, whereas a negative control anti-DNP F(ab')2 had no effect. CONCLUSIONS: MRL/lpr lupus-prone mice show exaggerated ICAM-1-dependent leucocyte-endothelial interactions in response to TNF-alpha. Increased leucocyte-endothelial interactions due to endothelial priming could contribute to the clinical link between infection and flares of lupus disease activity.

Animals↗
PubMed2003

A snapshot of MDT working and patient mapping in the UK colorectal cancer centres in 2002.

OBJECTIVES: To ascertain the position nationally of Colorectal Multi-Disciplinary Team (MDT) implementation as part of the NHS cancer plan. Also to define nationally patterns of 'bottlenecks' in the patient journey from referral to treatment. METHODS: A simple questionnaire was sent to the lead clinician at all 183 cancer networks asking about their current MDT organization, and whether they had undertaken a mapping exercise of the patient journey. RESULTS: Ninety-one percent (n = 166) of the questionnaires were returned, and of these 90% (n = 150) stated that their MDT meeting was up and running. Sixty-two percent (n = 102) stated they were having problems running their MDT meeting and of these 32% (n = 33) did not have a dedicated MDT clerk. Several of the Calman-Hine identified 'core personnel' are attending infrequently. Sixty-four percent (n = 107) of respondents have carried out a patient mapping process with 65% (n = 70) claiming it was a success, leaving 27% (n = 32) with no immediate plans to undertake the process. CONCLUSIONS: Although MDT activity is near-universal, the survey has highlighted a wide variation in MDT meeting implementation across England, with the same problems being experienced by most centres. Organizational problems are common, and we feel that a dedicated MDT clerk is essential for smooth running. Further areas that require development have been identified by most respondents as radiological, oncological and endoscopic services. It is anticipated that true collaboration nationally will develop, and contact with the nine pilot sites is encouraged to explore solutions to difficulties.

Cancer Care Facilities↗
PubMed2002

In vitro behaviour of human osteoblasts on dentin and bone.

This investigation studied how the behaviour of isolated osteoblasts on standard tissue culture polystyrene compared with cells cultured on cut surfaces of dentin, a natural calcified material. Cellular attachment, viability and growth were monitored in parallel cultures of human osteosarcoma cell lines (MG63, HOS TE85, SaOS-2) and primary human osteoblast-like cells (HOBs). Culture plastic was either left untreated or roughened with abrasive paper of various grit sizes (4000-1200 grit) in order to obtain a level of roughness comparable to that of the dentin slices. Cell counting and intracellular BCECF staining showed that after an initial incubation of 2 h, the primary cells attached and spread out more quickly on the different substrates than the three cell lines. The primary cells also showed a stronger mitochondrial staining and viability on dentin. During subsequent culture morphological differences appeared with the cells on dentin displaying more cellular extensions. All three cell lines proliferated more slowly on dentin than on plastic. In contrast, the primary HOBs were not significantly affected in their growth by the different substrates. Total and specific alkaline phosphatase (AP) activity of the cell lines was not significantly affected by the different substrates after short-term adhesion, but it was increased for the primary cells on the dentin. However, after 2-3 days of culture, AP was decreased on the dentin slices for both the cell lines and primary HOBs. Plasma treatment of the roughened plastic did not alter cellular viability or AP activity, suggesting that grinding of the surface did not affect the property of the culture plastic to support cell attachment and growth. In conclusion, the results show that not only do osteoblastic cells behave differently on a natural calcified substrate surface than on standard culture plastic, but also that differences were evident between the various cell types, in particular the primary HOB versus the continuous cell lines.

Cell Adhesion↗
PubMed2002

A second look at anorectal infections in cancer patients in a large cancer institute: the success of early intervention with antibiotics and surgery.

BACKGROUND: Infection of anorectal region represents a significant complication of anti-cancer therapy. Anorectal infection occurs in patients receiving aggressive chemotherapy. Untreated infection leads to substantial morbidity and in the past, mortality. METHODS: 82 episodes of anorectal infection in 64 patients with malignant diseases occurring over 12 years at the National Cancer Institute (NCI) were retrospectively reviewed. RESULTS: The overall incidence is comparable to the prior NCI experience despite a shift in patient population to a lower percentage of lymphoid/leukemic diagnoses (34% vs 77%). There were no deaths associated with anorectal infection in the 12 years reviewed compared to seven of 44 in the previous decade (p = 0.003). Antibiotic therapy alone was successful in managing 25/82. Only five episodes were treated with surgery alone compared to nearly 45% in the previous decade. There were no major surgical complications. Neutropenia was present in 43/82 episodes. 11 episodes were complicated by bacteremia, predominately with Staphylococcus non- aureus (n = 8). Wound cultures were performed in 36 episodes from 23 patients yielding 99 separate isolates. Gram-negative isolates were most common overall. Modification of antibiotic therapy with further anaerobic coverage was administered successfully in 39/77 episodes. CONCLUSION: This study illustrates that anorectal infections in cancer patients can be successfully managed with antibiotic therapy and local care; surgery can be withheld unless there is evidence of progressive infection or substantial fluctuance and necrosis.

Adolescent↗
PubMed2002

The effect of torsion on the appearance of the rupture surface of the ACL of rabbits.

This study investigates how torsion affects the mechanical properties and appearance of the rupture surfaces of the anterior cruciate ligament (ACL). Rupture of the ACL is frequent among athletes and twisting of the knee during rupture is often implicated as a major factor in the injury. The problem is compounded by the ACL having limited ability to heal when ruptured. Thirty-two specimens were stretched to failure at tension--torsion rates of 10 mm/min and 1 degree/s, 10 mm/min and 40 degree/s, 500 mm/min and 1 degree/s and 500 mm/min and 40 degree/s. Results from these experiments were compared with those from pure tension tests performed at displacement rates of 10 and 500 mm/min. The changes that occurred during the rupture process were monitored by video recordings and the appearance of the rupture surfaces studied by scanning electron microscopy (SEM). We found significant differences in stiffness between tests carried out with and without torsion. The macro and microscopic appearance of the ruptures from specimens tested with torsion at different displacement rates were similar although, for the slow displacement rate, the rupture surfaces were different from those from tests carried out without torsion. The failure modes for all tests carried with torsion were similar, with all the specimens failing in the ligament substance by fibre pullout. The modes of failure for tests carried at 500 mm/min with torsion were different from those tested without torsion. Half of the specimens tested without torsion failed by tibial avulsion, whereas all specimens tested with torsion failed in the ligament substance. These results suggest that torsion affects both mechanical and structural properties of the ACL and should, therefore, be considered when designing or selecting replacements for the ACL.

Animals↗
PubMed2001

Differentially expressed late constituents of the epidermal cornified envelope.

Barrier activity of skin and internal barrier-forming epithelial linings are conferred by a lipid-corneocyte structure (stratum corneum in skin). The integrity of the corneocytes depends on the outer cornified envelope and is essential for maintenance of barrier function. During epidermal development and differentiation, proteins are sequentially incorporated into the envelope via action of epidermal transglutaminases in a well documented process. However, recent knockouts of major cornified envelope constituents have failed to disrupt barrier function significantly, suggesting that additional unidentified components are involved. We report a new gene cluster in the epidermal differentiation complex at human 1q21 encoding a family of 18 proteins that are substrates for epidermal transglutaminases. These proteins incorporate into the cornified envelope late in development and late in the process of envelope maturation during epidermal differentiation. The genes cluster within the epidermal differentiation complex according to expression pattern, i.e., epidermally expressed proteins cluster together while proteins from internal barrier-forming epithelia also cluster. We propose that these proteins modulate barrier activity over the surface of the animal, in a manner analogous to that proposed for the well characterized cornified envelope precursors, the small proline-rich proteins. To emphasize the incorporation of these proteins late in envelope assembly, we call the human proteins late envelope proteins.

Animals↗
PubMed2001

Identification of a novel phosphonocarboxylate inhibitor of Rab geranylgeranyl transferase that specifically prevents Rab prenylation in osteoclasts and macrophages.

Nitrogen-containing bisphosphonate drugs inhibit bone resorption by inhibiting FPP synthase and thereby preventing the synthesis of isoprenoid lipids required for protein prenylation in bone-resorbing osteoclasts. NE10790 is a phosphonocarboxylate analogue of the potent bisphosphonate risedronate and is a weak anti-resorptive agent. Although NE10790 was a poor inhibitor of FPP synthase, it did inhibit prenylation in J774 macrophages and osteoclasts, but only of proteins of molecular mass approximately 22-26 kDa, the prenylation of which was not affected by peptidomimetic inhibitors of either farnesyl transferase (FTI-277) or geranylgeranyl transferase I (GGTI-298). These 22-26-kDa proteins were shown to be geranylgeranylated by labelling J774 cells with [(3)H]geranylgeraniol. Furthermore, NE10790 inhibited incorporation of [(14)C]mevalonic acid into Rab6, but not into H-Ras or Rap1, proteins that are modified by FTase and GGTase I, respectively. These data demonstrate that NE10790 selectively prevents Rab prenylation in intact cells. In accord, NE10790 inhibited the activity of recombinant Rab GGTase in vitro, but did not affect the activity of recombinant FTase or GGTase I. NE10790 therefore appears to be the first specific inhibitor of Rab GGTase to be identified. In contrast to risedronate, NE10790 inhibited bone resorption in vitro without markedly affecting osteoclast number or the F-actin "ring" structure in polarized osteoclasts. However, NE10790 did alter osteoclast morphology, causing the formation of large intracellular vacuoles and protrusion of the basolateral membrane into large, "domed" structures that lacked microvilli. The anti-resorptive activity of NE10790 is thus likely due to disruption of Rab-dependent intracellular membrane trafficking in osteoclasts.

Alkyl and Aryl Transferases↗
PubMed2001

Specific packaging of nodaviral RNA2 requires the N-terminus of the capsid protein.

Flock house virus (FHV), a member of the family Nodaviridae, is a nonenveloped, icosahedral insect virus whose capsids are assembled from 180 copies of a single type of coat protein. The viral genome is split between two segments of single-stranded positive-sense RNA, RNA1 and RNA2, which are packaged into a single virion. We previously demonstrated that synthesis of FHV coat protein in the baculovirus expression system results in assembly of virus-like particles whose capsids are indistinguishable from those of native virions, although the encapsidated RNA represents primarily cellular RNA. In contrast, expression of a deletion mutant lacking N-terminal residues 2-31 results in formation of multiple types of particles which differ in size, shape, and RNA contents. We postulated that the polymorphism was imposed by the type of RNA that the coat protein selected for packaging. In the current study we tested this hypothesis by analyzing the assembly of the mutant coat protein in Drosophila cells in the presence of replicating FHV RNAs. As anticipated, the resulting particles had the same shape and dimensions as wt virions. Surprisingly, however, they contained little RNA2 while packaging of RNA1 was not affected. Small amounts of defective interfering RNAs, which emerged rapidly in the presence of the mutant coat protein, were also detected. Taken together, these observations confirm our earlier hypothesis that selection of nonviral RNAs for packaging can significantly alter the assembly process. In addition, they demonstrate that the N-terminus of the FHV coat protein contains important determinants for recognition and packaging of RNA2. Our results provide the first evidence that encapsidation of the two genomic RNAs occurs independently and that the coat protein uses different regions for the recognition of RNA1 and RNA2.

Amino Acid Sequence↗
PubMed2001

Viral capsid mobility: a dynamic conduit for inactivation.

Mass spectrometry and fluorescent probes have provided direct evidence that alkylating agents permeate the protein capsid of naked viruses and chemically inactivate the nucleic acid. N-acetyl-aziridine and a fluorescent alkylating agent, dansyl sulfonate aziridine, inactivated three different viruses, flock house virus, human rhinovirus-14, and foot and mouth disease virus. Mass spectral studies as well as fluorescent probes showed that alkylation of the genome was the mechanism of inactivation. Because particle integrity was not affected by selective alkylation (as shown by electron microscopy and sucrose gradient experiments), it was reasoned that the dynamic nature of the viral capsid acts as a conduit to the interior of the particle. Potential applications include fluorescent labeling for imaging viral genomes in living cells, the sterilization of blood products, vaccine development, and viral inactivation in vivo.

Animals↗
PubMed2001

Potential cost-effectiveness of one-time screening for lung cancer (LC) in a high risk cohort.

The development of low-dose helical computed-tomography (CT) scanning to detect nodules as small as a few mm has sparked renewed interest in lung cancer (LC) screening. The objective of this study was to assess the potential health effects and cost-effectiveness of a one-time low-dose helical CT scan to screen for LC. We created a decision analysis model using baseline results from the Early Lung Cancer Action Project (ELCAP); Surveillance, Epidemiology and End Results (SEER) registry public-use database; screening program costs estimated from 1999 Medicare reimbursement rates; and annual costs of managing cancer and non-cancer patients from Riley et al. (1995) [Med Care 1995;33(8):828-841] and Taplin et al. (1995) [J Natl Cancer Inst 1995;87(6):417-26]. The main outcome measures included years of life, cost estimates of baseline diagnostic screening and follow up, and cost-effectiveness of screening. We found that in a very high-risk cohort (LC prevalence of 2.7%) of patients between 60 and 74 years of age, a one-time screen appears to be cost-effective at $5940 per life year saved. In a lower risk general population of smokers (LC prevalence of 0.7%), a one-time screen appears to be cost-effective at $23100 per life year. Even when a lead-time bias of 1 year is incorporated into the model for a low risk population, the cost-effectiveness is estimated at $58183 per life year. Based on the assumptions embedded in this model, one-time screening of elderly high-risk patients for LC appears to be cost-effective.

Aged↗
PubMed2001

Preliminary comparison of the rupture of human and rabbit anterior cruciate ligaments.

OBJECTIVE: This study was aimed at examining ruptures of the human anterior cruciate ligaments by scanning electron microscopy and video imaging and comparing the appearance of the rupture surfaces with those from rabbit anterior cruciate ligaments. DESIGN: The specimens were tested to failure as femur-anterior cruciate ligament-tibia complexes using an Instron 8511 materials testing machine. BACKGROUND: Rupture of the anterior cruciate ligament is a major clinical problem, leading to instability of the knee joint. Due to the frequency and potential severity of the injuries, a need still exists for information on the biomechanical properties of ligaments under loading conditions, which occur at the time of trauma. METHODS: Four human femur-anterior cruciate ligament-tibia complexes were loaded to failure at a displacement rate of 0.008 m/s. Video recordings of the tests were used to study the progress of the ruptures and to compare the modes of failure of the ligaments. Scanning electron microscopy was employed to study the appearance of collagen fibres at the rupture surfaces. RESULTS: The modes of failure of the rabbit anterior cruciate ligament and appearance of the rupture surfaces were similar to those of the human anterior cruciate ligaments. CONCLUSION: The rabbit anterior cruciate ligament provides a model for investigating failure of the human ligament during trauma. RELEVANCE: The results will be of significance since most studies on ligaments are carried out on animal models with the intention of applying the deductions from the results to human ligaments. Examining the appearance of collagen fibres at these surfaces may help us to understand more about what actually happens during and after ligament rupture.

Animals↗