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1.
Lavanya Rishishwar Lee S. Katz Nitya V. Sharma Lori Rowe Michael Frace Jennifer Dolan Thomas Brian H. Harcourt Leonard W. Mayer I. King Jordan 《Journal of bacteriology》2012,194(20):5649-5656
Containment strategies for outbreaks of invasive Neisseria meningitidis disease are informed by serogroup assays that characterize the polysaccharide capsule. We sought to uncover the genomic basis of conflicting serogroup assay results for an isolate () from a patient with acute meningococcal disease. To this end, we characterized the complete genome sequence of the M16917 isolate and performed a variety of comparative sequence analyses against N. meningitidis reference genome sequences of known serogroups. Multilocus sequence typing and whole-genome sequence comparison revealed that M16917 is a member of the ST-11 sequence group, which is most often associated with serogroup C. However, sequence similarity comparisons and phylogenetic analysis showed that the serogroup diagnostic capsule polymerase gene (synD) of M16917 belongs to serogroup B. These results suggest that a capsule-switching event occurred based on homologous recombination at or around the capsule locus of M16917. Detailed analysis of this locus uncovered the locations of recombination breakpoints in the M16917 genome sequence, which led to the introduction of an ∼2-kb serogroup B sequence cassette into the serogroup C genomic background. Since there is no currently available vaccine for serogroup B strains of N. meningitidis, this kind capsule-switching event could have public health relevance as a vaccine escape mutant. M16917相似文献
2.
Streptococcus suis causes meningitis and other serious infections in pigs and humans, and binds to host cell globotriosylceramide. In order to determine the essential hydroxyls involved in binding, the complete set of monodeoxy derivatives of the receptor trisaccharide Gal1-Gal1-4Glc were tested as inhibitors of bacterial hemagglutination. Removal of the 4-, 6, 2 or 3-hydroxyls abolished inhibitory activity, which indicated that they were critically involved in binding. The same results were obtained using synthetic lipid-linked monodeoxy derivatives of the trisaccharides in a thin-layer overlay assay. The PN and PO subtypes of the S. suis adhesin showed similar binding patterns. The hydroxyls of the glucose moiety were not critical for binding, although the adhesin binds better to the trisaccharide Gal1-4Gal1-4Glc than the disaccharide Gal1-4Gal. 相似文献
3.
Summary Determinations of current-voltage relationships are widely employed in the characterization of epithelial sodium transport. In order to determine the protocol dependence of transport parameters in the toad urinary bladder, studies were carried out in the presence and absence of amiloride, an inhibitor of active sodium transport. With symmetric positive and negative perturbations of the transepithelial electrical potential difference (0±100 mV) for 30 sec, the amiloride-sensitive current-voltage (i
a
-) relationship was near linear over the range –75+100 mV, indicating constancy of the conductance a and the apparent electromotive force E
Na, lumped parameters of the standard electrical equivalent circuit model of the active transport system. With a reverse protocol (±1000 mV) or 15 min perturbations thei
a
- relationships were highly nonlinear. Nonlinearity reflected voltage dependence of parameters: perturbations that increased active transport decreased E
Na and increased a, as evaluated from 10 sec perturbations of ; slowing of active transport produced the converse changes. These effects are usefully analyzed in both quasi-steady states and true steady states by means of a detailed equivalent circuit incorporating the significant ionic currents across each plasma membrane. Precise understanding of the significance of a and E
Na will require characterization of the partial ionic conductances on perturbation of . 相似文献
4.
Charles B. Olson 《Origins of life and evolution of the biosphere》1981,11(4):353-368
Life on Earth is essentially nucleic acids (NAs) influencing peptide synthesis such that NA replication is favored. It is proposed that the ability to synthesize polypeptides evolved gradually — one peptide bond at a time. The proposed evolution of the peptide synthesis apparatus begins with a transfer NA (tNA) which catalyzes the transfer of activated amino acids to accessible amino groups in its environment. The resulting capped molecules (with single amino acid caps) in turn favor NA replication. The proposed evolution of the peptide synthesis apparatus from the tNA onward is characterized by a progressive increase in the number of amino acids per cap: two tNAs jointly produce a dipeptide cap, three tNAs jointly produce a tripeptide cap, etc. Messenger NAs evolve because they can specify the composition and sequence order of the peptide caps. Lastly, ribosomal NAs evolve. The origin, expansion, and standardization of the genetic code are discussed. It is proposed that the present triplet code evolved by a process of codon length refinement, and that originally codons of varying lengths were allowable, as were unassigned bases between codons. An environmental supply of activated compounds for early evolving entities is proposed. An environmental NA replication process via single template-directed bond formation events is proposed. An environmental retention and redistribution process is proposed to have acted as a functional substitute for the cell wall and cell division of early evolving entities. 相似文献
5.
Pablo González-Jara Aurora Fraile Tomás Canto Fernando García-Arenal 《Journal of virology》2009,83(15):7487-7494
The multiplicity of infection (MOI), i.e., the number of virus genomes that infect a cell, is a key parameter in virus evolution, as it determines processes such as genetic exchange among genomes, selection intensity on viral genes, epistatic interactions, and the evolution of multipartite viruses. In fact, the MOI level is equivalent to the virus ploidy during genome expression. Nevertheless, there are few experimental estimates of MOI, particularly for viruses with eukaryotic hosts. Here we estimate the MOI of Tobacco mosaic virus (TMV) in its systemic host, Nicotiana benthamiana. The progress of infection of two TMV genotypes, differently tagged with the green or red fluorescent proteins GFP and RFP, was monitored by determining the number of leaf cell protoplasts that showed GFP, RFP, or GFP and RFP fluorescence at different times postinoculation. This approach allowed the quantitative analysis of the kinetics of infection and estimation of the generation time and the number of infection cycles required for leaf colonization. MOI levels were estimated from the frequency of cells infected by only TMV-GFP or TMV-RFP. The MOI was high, but it changed during the infection process, decreasing from an initial level of about 6 to a final one of 1 to 2, with most infection cycles occurring at the higher MOI levels. The decreasing MOI can be explained by mechanisms limiting superinfection and/or by genotype competition within double-infected cells, which was shown to occur in coinfected tobacco protoplasts. To our knowledge, this is the first estimate of MOI during virus colonization of a eukaryotic host.Virus evolution has been a very active area of research in the last few decades, as viruses are both important pathogens of humans, animals, and plants and good models to experimentally test hypotheses on parasite evolution or, more generally, central questions on evolutionary biology (11, 12, 21, 36). Considerable efforts have been devoted to modeling the evolution of viral populations. However, contrasting the theoretical models with reality may be hindered by limited experimental information on important parameters of the virus life cycle. The multiplicity of infection (MOI), i.e., the number of virus particles or genomes that may infect a cell, is a key parameter in many models of virus evolution (5, 6, 14, 15, 37, 38, 39, 52, 53, 57, 61) for which experimental estimates are scant.When a cell is coinfected by different viral genomes, competition may lead to decreased fitness of individual genotypes in comparison with their fitness in single infections (15, 31, 40). Thus, limiting coinfection may result in a selective advantage for viruses (58), which have developed mechanisms to prevent superinfection of previously infected cells (51, 60). On the other hand, infection of a cell by more than one virus genome is a prerequisite for two central phenomena in virus genetics to take place: recombination and complementation of defective mutants. Recombination between viral strains during replication in the same cell and complementation of defective mutants have been extensively documented for viruses infecting prokaryotes, animals, and plants (2, 25, 56), indicating that there must be some degree of coinfection and, hence, that the MOI must be higher than one in at least some infected cells. However, estimates of MOI in the natural hosts of viruses are surprisingly scarce in spite of this parameter''s relevance: values of about 2 to 3 have been reported for different DNA or RNA bacteriophages (26, 41, 51, 58), and a value of 4 to 5 was reported for Autographa californica nuclear polyhedrosis virus infecting larvae of the moth Tricoplusia ni (3), to our knowledge, the only estimate for a virus in its eukaryotic host. We are not aware of estimates reported for viruses infecting mammals or plants, although a MOI of about 3 can be inferred from the number of proviral copies of HIV in spleen cells of infected patients (29). This paucity of data may be due to the technical difficulty of directly measuring MOI, particularly within a eukaryotic host. Genetic approaches may provide valid alternatives for estimating MOI levels (3, 58), and here, the MOI of a plant virus is estimated through the analysis of the relative frequencies of two genotypes during the process of host colonization.Host colonization by plant-infecting viruses has been known for a long time to be a two-step phenomenon. First, colonization proceeds slowly from the initially infected cells to their neighbors by way of the cytoplasmic connections called plasmodesmata, a process known as cell-to-cell movement. After infection thus reaches the cells in the vasculature, the second step, known as long-distance or systemic movement, occurs as viruses move faster to distant organs through the vascular tissue, the phloem in most cases (59). As a result of these processes, the virus population within the infected plant may be strongly structured. Analyses of different viruses in different host plant species have shown that systemic movement causes population bottlenecks that may be severe (16, 28, 32, 34, 46), resulting in differences in the genetic composition of the virus subpopulations in different systemically infected organs. No analysis of population bottlenecks during cell-to-cell movement has been reported, but data indicate that the virus population within a leaf has a strong spatial structure with a separate distribution of different genotypes in different leaf areas. These reports derive from analyses of viruses that differ in genomic organization and gene expression strategies in different host plant species (9, 10, 23, 55); they indicate that a separate distribution of viral genotypes within the infected leaf is a general phenomenon and suggest limitation of coinfection. Data on the spatial exclusion of virus genotypes within the infected leaf are in apparent contradiction with the abundant evidence of recombination and complementation of defective mutants, which has been widely documented for plant viruses (19, 44, 50, 62). It should be pointed out that all reports on the spatial exclusion of virus genotypes in an infected leaf derive from microscopy observations, mostly at late times after infection of the tissue. No information is available on the kinetics of leaf colonization by viruses, and current data do not allow the estimation of MOI.In this report, we estimate the MOI of a plant RNA virus, Tobacco mosaic virus (TMV), in its systemic host, Nicotiana benthamiana. For this, we have reexamined the process of virus colonization by monitoring the progress of infection of two TMV genotypes in inoculated and in systemically infected leaves. The two TMV genotypes differed in the expression of fluorescent tags, either the green fluorescent protein (GFP) from Aequorea victoria (42, 43) or a red fluorescent protein (RFP) from Discosoma sp. (49). The expression of GFP and RFP allowed the precise quantification of the number of cells infected by either one or both TMV genotypes, and these data allowed the estimation of genotype frequencies and of MOI. The results show evidence of strong spatial structure of the virus population, with most cells being infected by either TMV-GFP or TMV-RFP alone and only a small fraction of cells being double infected. The kinetics of the single and double infections show that the MOI changes with time, decreasing as colonization progresses and therefore suggesting that exclusion mechanisms operate at later times after infection. 相似文献
6.
The primary structures of three -amylase inhibitors (TAI, DAI, and MAI-2) consisting of glycoprotein subunits and from the respective seeds of three cultivars of Phaseolus beans, Toramame (Phaseolus vulgaris L.), Daifukumame (Phaseolus vulgaris L.), and Murasakihanamame (Phaseolus coccineus L.) were determined by sequencing the peptide fragments derived from their enzymatic digestions. Major sugar chains of the inhibitors were also assessed by analyzing glycopeptides in the enzymatic digests. The subunits, and , were shown to be composed of 76 and 139 amino acid residues, respectively, in each inhibitor. The overall amino acid sequences of the inhibitors were slightly different from one another. Furthermore, the sequence of TAI was the same as that deduced from a cDNA clone encording -amylase inhibitor-1 from the common bean (Phaseolus vulgaris L.). It was also revealed that there were two N-glycosylation sites in each -subunit: PA-derivatives of the major N-glycans were estimated to be M6B at Asn(12) and M9A at Asn(65). Each -subunit of TAI and MAI-2 had two N-glycosylation sites, while the -subunit of DAI had only one site. The major N-glycans pyridylaminated were estimated to be M3X at Asn(63) in each -subunit and M3FX at Asn(83) in -subunits of TAI and MAI-2. 相似文献
7.
Rudolf I. Salganik Rahmet I. Bersimbaev Svetlana V. Argutinskaya Elena V. Kiseleva Ninel B. Khristolyubova Victoria I. Deribas 《Molecular and cellular biochemistry》1976,12(3):181-191
Summary The regulation patterns of gastric acid secretion in rats were investigated. Pentagastrin and histamine stimulate gastric acid secretion, but the inhibitors of DNA-dependent synthesis of RNA and of proteins prevent only the pentagastrin action. It has been found that pentagastrin induces histidine decarboxylase in gastric mucosa, ensuring local accumulation of histamine. The latter activates adenylate cyclase and results in 3,5-AMP accumulation in gastric tissues. The administration of pentagastrin, histamine or 3,5-AMP enhances the activity of gastric carbonic anhydrase, the enzyme which takes part in HCI formation. The data suggest that these three compounds act sequentially (pentagastrin histamine 3,5-AMP) and the effect of the last one could be mediated through 3,5-AMP dependent protein kinase. The experiments in vitro demonstrated that gastric carbonic anhydrase can be separated into two isoenzymes and the phosphorylation of one of them by the 3,5-AMP dependent protein kinase sharply increases its activity. The findings raise the possibility that histamine and 3,5-AMP, mediating gastrin action, form together with enzymes (histidine decarboxylase, adenylate cyclase, protein kinase, carbonic anhydrase) a cascade of amplifiers.Autoradiographic studies have shown that [3H]-pentagastrin is not bound by oxyntic cells but adheres preferentially to histamine-producing-like endocrine cells and to the chief cells, while3H-histamine adheres preferentially to oxyntic and to chief cells. Electron microscopy indicates that only pentagastrin (but not histamine) initiates in-like endocrine cells ultrastructural changes characteristic for induction. Pentagastrin, histamine and 3,5-AMP administration produces in oxyntic cells ultrastructural changes typical for the secretion processes.These results lead to assumption that pentagastrin (gastrin) induces histidine decarboxylase in-like endocrine cells of gastric glands. Histamine which is secreted enhances adenylate cyclase activity in the neighbouring oxyntic cells where 3,5-AMP dependent protein kinase activates carbonic anhydrase by means of phosphorylation. These different cells form, probably, a multicellular functional unit for gastric acid secretion.An invited article. 相似文献
8.
Ching Leang Xinlei Qian Tünde Mester Derek R. Lovley 《Applied and environmental microbiology》2010,76(12):4080-4084
Immunogold localization revealed that OmcS, a cytochrome that is required for Fe(III) oxide reduction by Geobacter sulfurreducens, was localized along the pili. The apparent spacing between OmcS molecules suggests that OmcS facilitates electron transfer from pili to Fe(III) oxides rather than promoting electron conduction along the length of the pili.There are multiple competing/complementary models for extracellular electron transfer in Fe(III)- and electrode-reducing microorganisms (8, 18, 20, 44). Which mechanisms prevail in different microorganisms or environmental conditions may greatly influence which microorganisms compete most successfully in sedimentary environments or on the surfaces of electrodes and can impact practical decisions on the best strategies to promote Fe(III) reduction for bioremediation applications (18, 19) or to enhance the power output of microbial fuel cells (18, 21).The three most commonly considered mechanisms for electron transfer to extracellular electron acceptors are (i) direct contact between redox-active proteins on the outer surfaces of the cells and the electron acceptor, (ii) electron transfer via soluble electron shuttling molecules, and (iii) the conduction of electrons along pili or other filamentous structures. Evidence for the first mechanism includes the necessity for direct cell-Fe(III) oxide contact in Geobacter species (34) and the finding that intensively studied Fe(III)- and electrode-reducing microorganisms, such as Geobacter sulfurreducens and Shewanella oneidensis MR-1, display redox-active proteins on their outer cell surfaces that could have access to extracellular electron acceptors (1, 2, 12, 15, 27, 28, 31-33). Deletion of the genes for these proteins often inhibits Fe(III) reduction (1, 4, 7, 15, 17, 28, 40) and electron transfer to electrodes (5, 7, 11, 33). In some instances, these proteins have been purified and shown to have the capacity to reduce Fe(III) and other potential electron acceptors in vitro (10, 13, 29, 38, 42, 43, 48, 49).Evidence for the second mechanism includes the ability of some microorganisms to reduce Fe(III) that they cannot directly contact, which can be associated with the accumulation of soluble substances that can promote electron shuttling (17, 22, 26, 35, 36, 47). In microbial fuel cell studies, an abundance of planktonic cells and/or the loss of current-producing capacity when the medium is replaced is consistent with the presence of an electron shuttle (3, 14, 26). Furthermore, a soluble electron shuttle is the most likely explanation for the electrochemical signatures of some microorganisms growing on an electrode surface (26, 46).Evidence for the third mechanism is more circumstantial (19). Filaments that have conductive properties have been identified in Shewanella (7) and Geobacter (41) species. To date, conductance has been measured only across the diameter of the filaments, not along the length. The evidence that the conductive filaments were involved in extracellular electron transfer in Shewanella was the finding that deletion of the genes for the c-type cytochromes OmcA and MtrC, which are necessary for extracellular electron transfer, resulted in nonconductive filaments, suggesting that the cytochromes were associated with the filaments (7). However, subsequent studies specifically designed to localize these cytochromes revealed that, although the cytochromes were extracellular, they were attached to the cells or in the exopolymeric matrix and not aligned along the pili (24, 25, 30, 40, 43). Subsequent reviews of electron transfer to Fe(III) in Shewanella oneidensis (44, 45) appear to have dropped the nanowire concept and focused on the first and second mechanisms.Geobacter sulfurreducens has a number of c-type cytochromes (15, 28) and multicopper proteins (12, 27) that have been demonstrated or proposed to be on the outer cell surface and are essential for extracellular electron transfer. Immunolocalization and proteolysis studies demonstrated that the cytochrome OmcB, which is essential for optimal Fe(III) reduction (15) and highly expressed during growth on electrodes (33), is embedded in the outer membrane (39), whereas the multicopper protein OmpB, which is also required for Fe(III) oxide reduction (27), is exposed on the outer cell surface (39).OmcS is one of the most abundant cytochromes that can readily be sheared from the outer surfaces of G. sulfurreducens cells (28). It is essential for the reduction of Fe(III) oxide (28) and for electron transfer to electrodes under some conditions (11). Therefore, the localization of this important protein was further investigated. 相似文献
9.
Characterization of RIPK3-mediated phosphorylation of the activation loop of MLKL during necroptosis
D A Rodriguez R Weinlich S Brown C Guy P Fitzgerald C P Dillon A Oberst G Quarato J Low J G Cripps T Chen D R Green 《Cell death and differentiation》2016,23(1):76-88
Mixed lineage kinase domain-like pseudokinase (MLKL) mediates necroptosis by translocating to the plasma membrane and inducing its rupture. The activation of MLKL occurs in a multimolecular complex (the ‘necrosome''), which is comprised of MLKL, receptor-interacting serine/threonine kinase (RIPK)-3 (RIPK3) and, in some cases, RIPK1. Within this complex, RIPK3 phosphorylates the activation loop of MLKL, promoting conformational changes and allowing the formation of MLKL oligomers, which migrate to the plasma membrane. Previous studies suggested that RIPK3 could phosphorylate the murine MLKL activation loop at Ser345, Ser347 and Thr349. Moreover, substitution of the Ser345 for an aspartic acid creates a constitutively active MLKL, independent of RIPK3 function. Here we examine the role of each of these residues and found that the phosphorylation of Ser345 is critical for RIPK3-mediated necroptosis, Ser347 has a minor accessory role and Thr349 seems to be irrelevant. We generated a specific monoclonal antibody to detect phospho-Ser345 in murine cells. Using this antibody, a series of MLKL mutants and a novel RIPK3 inhibitor, we demonstrate that the phosphorylation of Ser345 is not required for the interaction between RIPK3 and MLKL in the necrosome, but is essential for MLKL translocation, accumulation in the plasma membrane, and consequent necroptosis.Regulated necrotic cell death, or ‘necroptosis,'' is mediated by the interaction of activated receptor-interacting kinase-3 (RIPK3) and mixed lineage kinase like (MLKL).1, 2, 3 The function of RIPK3 to promote necroptosis can be induced by the activity of receptor-interacting protein kinase-1 (RIPK1),4 and is antagonized by the proteolytic activity of a complex formed by RIPK1, FADD, caspase-8 and c-FLIPL.5, 6, 7, 8, 9, 10 Inactive RIPK1 functions to inhibit RIPK3 activation, even under conditions in which RIPK3 is activated independently of RIPK1.11, 12, 13 These complex interactions help to account for the lethal effects of ablating FADD, caspase-8 or RIPK1.14MLKL is a substrate for RIPK3 kinase activity1, 2, 3 and appears to execute the process of necroptosis by targeting the plasma membrane.15, 16, 17 The phosphorylation of MLKL by RIPK3 has been proposed to promote necroptosis by inducing essential changes in the ‘latch'' of this pseudokinase, allowing the formation of oligomers, migration to plasma membrane15, 16, 17, 18 and binding to phosphatidylinositol lipids to directly disrupt membrane integrity.16, 19 Structurally, murine MLKL is composed of a pseudokinase domain (C-terminal region) and a four-helical bundle domain (4HBD) located in the N-terminal region.3, 20 The 4HBD domain is sufficient to oligomerize, bind to phosphatidylinositol lipids and trigger cell death.16, 19 However, the activation of full-length MLKL requires phosphorylation of residues in the activation loop in the pseudokinase domain. The residues Ser345, Ser347 and Thr349 within the murine MLKL activation loop are RIPK3 phosphorylation sites,3 corresponding to Thr357 and Ser358 in human MLKL.16 Upon RIPK3 phosphorylation, human MLKL shifts from its monomeric state to an active oligomeric state.16The residue Gln343 in the murine α-helix (residues Leu339 to Ser347) forms a hydrogen bond with Lys219 and the Ser345 and disruption of this coordinated state by phosphorylation of Ser345 has been proposed to destabilize the monomeric structure, promoting a conformational change in MLKL to an active state.3, 21 This hypothesis was supported by the specific mutations K219M, Q343A or S345D; all of which led to a form of MLKL form that promoted necroptosis independently of RIPK3.3, 16In this study, we examine serine and threonine residues within the activation loop of MLKL for their roles in necroptosis. We have developed an antibody anti-phospho-Ser345 and explore its use as a marker for necroptosis in murine cell systems. Using this antibody, together with described and novel inhibitors of RIPK3, we more fully explore the role of modifications in the active loop of MLKL during the process of necroptosis. 相似文献
10.
Sabine Drevet Bertrand Favier Emmanuel Brun Gaëtan Gavazzi Bernard Lardy 《Comparative medicine》2022,72(1):3
Osteoarthritis (OA) is a multidimensional health problem and a common chronic disease. It has a substantial impact on patient quality of life and is a common cause of pain and mobility issues in older adults. The functional limitations, lack of curative treatments, and cost to society all demonstrate the need for translational and clinical research. The use of OA models in mice is important for achieving a better understanding of the disease. Models with clinical relevance are needed to achieve 2 main goals: to assess the impact of the OA disease (pain and function) and to study the efficacy of potential treatments. However, few OA models include practical strategies for functional assessment of the mice. OA signs in mice incorporate complex interrelations between pain and dysfunction. The current review provides a comprehensive compilation of mouse models of OA and animal evaluations that include static and dynamic clinical assessment of the mice, merging evaluation of pain and function by using automatic and noninvasive techniques. These new techniques allow simultaneous recording of spontaneous activity from thousands of home cages and also monitor environment conditions. Technologies such as videography and computational approaches can also be used to improve pain assessment in rodents but these new tools must first be validated experimentally. An example of a new tool is the digital ventilated cage, which is an automated home-cage monitor that records spontaneous activity in the cages.Osteoarthritis (OA) is a multidimensional health problem and a common chronic disease.36 Functional limitations, the absence of curative treatments, and the considerable cost to society result in a substantial impact on quality of life.76 Historically, OA has been described as whole joint and whole peri-articular diseases and as a systemic comorbidity.9,111 OA consists of a disruption of articular joint cartilage homeostasis leading to a catabolic pathway characterized by chondrocyte degeneration and destruction of the extracellular matrix (ECM). Low-grade chronic systemic inflammation is also actively involved in the process.42,92 In clinical practice, mechanical pain, often accompanied by a functional decline, is the main reason for consultations. Recommendations to patients provide guidance for OA management.22, 33,49,86 Evidence-based consensus has led to a variety of pharmacologic and nonpharmacologic modalities that are intended to guide health care providers in managing symptomatic patients. Animal-based research is of tremendous importance for the study of early diagnosis and treatment, which are crucial to prevent the disease progression and provide better care to patients.The purpose of animal-based OA research is 2-fold: to assess the impact of the OA disease (pain and function) and to study the efficacy of a potential treatment.18,67 OA model species include large animals such as the horse, goat, sheep, and dog, whose size and anatomy are expected to better reflect human joint conditions. However, small animals such as guinea pig, rabbit, mouse, and rat represent 77% of the species used.1,87 In recent years, mice have become the most commonly used model for studying OA. Mice have several advantageous characteristics: a short development and life span, easy and low-cost breeding and maintenance, easy handling, small joints that allow histologic analysis of the whole joint,32 and the availability of genetically modified lines.108 Standardized housing, genetically defined strains and SPF animals reduce the genetic and interindividual acquired variability. Mice are considered the best vertebrate model in terms of monitoring and controlling environmental conditions.7,14,15,87 Mouse skeletal maturation is reached at 10 wk, which theoretically constitutes the minimal age at which mice should be entered into an OA study.64,87,102 However, many studies violate this limit by testing mice at 8 wk of age.Available models for OA include the following (32,111 physical activity and exercise induced OA; noninvasive mechanical loading (repetitive mild loading and single-impact injury); and surgically induced (meniscectomy models or anterior cruciate ligament transection). The specific model used would be based on the goal of the study.7 For example, OA pathophysiology, OA progression, and OA therapies studies could use spontaneous, genetic, surgical, or noninvasive models. In addition, pain studies could use chemical models. Lastly, post-traumatic studies would use surgical or noninvasive models; the most frequently used method is currently destabilization of the medial meniscus,32 which involves transection of the medial meniscotibial ligament, thereby destabilizing the joint and causing instability-driven OA. An important caveat for mouse models is that the mouse and human knee differ in terms of joint size, joint biomechanics, and histologic characteristics (layers, cellularity),32,64 and joint differences could confound clinical translation.10 Table 1. Mouse models of osteoarthritis.
Open in a separate windowSince all animal models have strengths and weaknesses, it is often best to plan using a number of models and techniques together to combine the results.In humans, the lack of correlation between OA imaging assessment and clinical signs highlights the need to consider the functional data and the quality of life to personalize OA management. Clinical outcomes are needed to achieve 2 main goals: to assess the impact of the OA in terms of pain and function and to study the efficacy of treatments.65 Recent reviews offer few practical approaches to mouse functional assessment and novel approaches to OA models in mice.7,32,67,75,79,83,87, 100,120 This review will focus on static and dynamic clinical assessment of OA using automatic and noninvasive emerging techniques (Test name Techniques Kind of assessment Output Specific equipment required Static measurement Von Frey filament testing Calibrated nylon filaments of various thickness (and applied force) are pressed against the skin of the plantar surface of the paw in ascending order of force Stimulus- evoked pain-like behavior
Mechanical stimuli - Tactile allodynia
The most commonly used test Latency to paw withdrawal
and
Force exerted are recorded Yes Knee extension test Apply a knee extension on both the intact and affected knee
or
Passive extension range of the operated knee joint under anesthesia Stimulus-evoked pain-like behavior Number of vocalizations evoked in 5 extensions None Hotplate Mouse placed on hotplate. A cutoff latency has been determined to avoid lesions Stimulus-evoked pain-like behavior
Heat stimuli- thermal sensitivity Latency of paw withdrawal Yes Righting ability Mouse placed on its back Neuromuscular screening Latency to regain its footing None Cotton swab test Bringing a cotton swab into contact with eyelashes, pinna, and whiskers Stimulus-evoked pain-like behavior
Neuromuscular screening Withdrawal or twitching response None Spontaneous activity Spontaneous cage activity One by one the cages must be laid out in a specific platform Spontaneous pain behavior
Nonstimulus evoked pain
Activity Vibrations evoked by animal movements Yes Open field analysis Experiment is performed in a clear chamber and mice can freely explore Spontaneous pain behavior
Nonstimulus evoked pain
Locomotor analysis Paw print assessment
Distance traveled, average walking speed, rest time, rearing Yes Gait analysis Mouse is placed in a specific cage equipped with a fluorescent tube and a glass plate allowing an automated quantitative gait analysis Nonstimulus evoked pain
Gait analysis
Indirect nociception Intensity of the paw contact area, velocity, stride frequency, length, symmetry, step width Yes Dynamic weight bearing system Mouse placed is a specific cage. This method is a computerized capacitance meter (similar to gait analysis) Nonstimulus evoked pain
Weight-bearing deficits
Indirect nociception Body weight redistribution to a portion of the paw surface Yes Voluntary wheel running Mouse placed is a specific cage with free access to stainless steel activity wheels. The wheel is connected to a computer that automatically record data Nonstimulus evoked pain
Activity Distance traveled in the wheel Yes Burrowing analysis Mouse placed is a specific cage equipped with steel tubes (32 cm in length and 10 cm in diameter) and quartz sand in Plexiglas cages (600 · 340x200 mm) Nonstimulus evoked pain
Activity Amount of sand burrowed Yes Digital video recordings Mouse placed is a specific cage according to the tool Nonstimulus evoked pain
Or
Evoked pain Scale of pain or specific outcome Yes Digital ventilated cage system Nondisrupting capacitive-based technique: records spontaneous activity 24/7, during both light and dark phases directly from the home cage rack Spontaneous pain behavior
Nonstimulus evoked pain
Activity-behavior Distance walked, average speed, occupation front, occupation rear, activation density.
Animal locomotion index, animal tracking distance, animal tracking speed, animal running wheel distance and speed or rotation Yes Challenged activity Rotarod test Gradual and continued acceleration of a rotating rod onto which mice are placed Motor coordination
Indirect nociception Rotarod latency: riding time and speed with a maximum cut off. Yes Hind limb and fore grip strength Mouse placed over a base plate in front of a connected grasping tool Muscle strength of limbs Peak force, time resistance Yes Wire hang analysis Suspension of the mouse on the wire and start the time Muscle strength of limbs: muscle function and coordination Latency to fall gripping None
(self -constructed)
Models | Pros | Cons | |
---|---|---|---|
Spontaneous | Wild type mice7,9,59,67,68,70,72,74,80,85,87,115,118,119,120 | - Model of aging phenotype - The less invasive model - Physiological relevance: mimics human pathogenesis - No need for technical expertise - No need for specific equipment | - Variability in incidence - Large number of animals at baseline - Long-term study: Time consuming (time of onset: 4 -15 mo) - Expensive (husbandry) |
Genetically modified mice2,7,25,40,50,52,67,72,79,80, 89,120 | - High incidence - Earlier time of onset: 18 wk - No need for specific equipment - Combination with other models | - Time consuming for the strain development - Expensive | |
Chemical- induced | Mono-iodoacetate injection7,11,46,47,60,66,90,91,101,128 | - Model of pain-like phenotype - To study mechanism of pain and antalgic drugs - Short-term study: Rapid progression (2-7 wk) - Reproducible - Low cost | - Need for technical expertise - Need for specific equipment - Systemic injection is lethal - Destructive effect: does not allow to study the early phase of pathogenesis |
Papain injection66,67,120 | - Short-term study: rapid progression - Low cost | - Need for technical expertise - Need for specific equipment - Does not mimic natural pathogenesis | |
Collagenase injection7,65,67,98 | - Short-term study: rapid progression (3 wk) - Low cost | - Need for technical expertise - Need for specific equipment - Does not mimic natural pathogenesis | |
Non-invasive | High-fat diet (Alimentary induced obesity model)5,8,43,45,57,96,124 | Model of metabolic phenotype No need for technical expertise No need for specific equipment Reproducible | Long-term study: Time consuming (8 wk–9 mo delay) Expensive |
Physical activity and exercise model45,73 | Model of post traumatic phenotype No need for technical expertise | Long-term study: time consuming (18 mo delay) Expensive Disparity of results | |
Mechanical loading models Repetitive mild loading models Single-impact injury model7,16,23,24, 32,35,104,105,106 | Model of post traumatic phenotype Allow to study OA development Time of onset: 8-10 wk post injury Noninvasive | Need for technical expertise Need for specific equipment Heterogeneity in protocol practices Repetitive anesthesia required or ethical issues | |
Surgical | Ovariectomy114 | Contested. | |
Meniscectomy model7,32,63,67,87 | Model of post traumatic phenotype High incidence Short-term study: early time of onset (4 wk from surgery) To study therapies | Need for technical expertise Need for specific equipment Surgical risks Rapid progression compared to human | |
Anterior cruciate ligament transection (ACLT)7,39,40,61,48,67,70,87,126 | Model of posttraumatic phenotype High incidence Short-term study: early time of onset (3-10 wk from surgery) Reproducible To study therapies | Need for technical expertise Need for specific equipment Surgical risks Rapid progression compared to human | |
Destabilization of medial meniscus (DMM)7,32,39,40 | Model of post traumatic phenotype High incidence Short-term study: early time of onset (4 wk from surgery) To study therapies The most frequently used method | Need for technical expertise Need for specific equipment Surgical risks Rapid progression compared to human |
Mechanical stimuli - Tactile allodynia
The most commonly used test
and
Force exerted are recorded
or
Passive extension range of the operated knee joint under anesthesia
Heat stimuli- thermal sensitivity
Neuromuscular screening
Nonstimulus evoked pain
Activity
Nonstimulus evoked pain
Locomotor analysis
Distance traveled, average walking speed, rest time, rearing
Gait analysis
Indirect nociception
Weight-bearing deficits
Indirect nociception
Activity
Activity
Or
Evoked pain
Nonstimulus evoked pain
Activity-behavior
Animal locomotion index, animal tracking distance, animal tracking speed, animal running wheel distance and speed or rotation
Indirect nociception
(self -constructed)