Stress-induced flowering

Many plant species can be induced to flower by responding to stress factors. The short-day plants Pharbitis nil and Perilla frutescens var. crispa flower under long days in response to the stress of poor nutrition or low-intensity light. Grafting experiments using two varieties of P. nil revealed that a transmissible flowering stimulus is involved in stress-induced flowering. The P. nil and P. frutescens plants that were induced to flower by stress reached anthesis, fruited and produced seeds. These seeds germinated, and the progeny of the stressed plants developed normally. Phenylalanine ammonialyase inhibitors inhibited this stress-induced flowering, and the inhibition was overcome by salicylic acid (SA), suggesting that there is an involvement of SA in stress-induced flowering. PnFT2, a P. nil ortholog of the flowering gene FLOWERING LOCUS T (FT) of Arabidopsis thaliana, was expressed when the P. nil plants were induced to flower under poor-nutrition stress conditions, but expression of PnFT1, another ortholog of FT, was not induced, suggesting that PnFT2 is involved in stress-induced flowering.Key words: flowering, stress, phenylalanine ammonia-lyase, salicylic acid, FLOWERING LOCUS T, Pharbitis nil, Perilla frutescensFlowering in many plant species is regulated by environmental factors, such as night-length in photoperiodic flowering and temperature in vernalization. On the other hand, a short-day (SD) plant such as Pharbitis nil (synonym Ipomoea nil) can be induced to flower under long days (LD) when grown under poor-nutrition, low-temperature or high-intensity light conditions.^1–9 The flowering induced by these conditions is accompanied by an increase in phenylalanine ammonia-lyase (PAL) activity.¹⁰ Taken together, these facts suggest that the flowering induced by these conditions might be regulated by a common mechanism. Poor nutrition, low temperature and high-intensity light can be regarded as stress factors, and PAL activity increases under these stress conditions.¹¹ Accordingly, we assumed that such LD flowering in P. nil might be induced by stress. Non-photoperiodic flowering has also been sporadically reported in several plant species other than P. nil, and a review of these studies suggested that most of the factors responsible for flowering could be regarded as stress. Some examples of these factors are summarized in 12^–14

Table 1

Some cases of stress-induced flowering

Natural Infection of Burkholderia pseudomallei in an Imported Pigtail Macaque (Macaca nemestrina) and Management of the Exposed Colony

Identification of the select agent Burkholderia pseudomallei in macaques imported into the United States is rare. A purpose-bred, 4.5-y-old pigtail macaque (Macaca nemestrina) imported from Southeast Asia was received from a commercial vendor at our facility in March 2012. After the initial acclimation period of 5 to 7 d, physical examination of the macaque revealed a subcutaneous abscess that surrounded the right stifle joint. The wound was treated and resolved over 3 mo. In August 2012, 2 mo after the stifle joint wound resolved, the macaque exhibited neurologic clinical signs. Postmortem microbiologic analysis revealed that the macaque was infected with B. pseudomallei. This case report describes the clinical evaluation of a B. pseudomallei-infected macaque, management and care of the potentially exposed colony of animals, and protocols established for the animal care staff that worked with the infected macaque and potentially exposed colony. This article also provides relevant information on addressing matters related to regulatory issues and risk management of potentially exposed animals and animal care staff.Abbreviations: CDC, Centers for Disease Control and Prevention; IHA, indirect hemagglutination assay; PEP, postexposure prophylacticBurkholderia pseudomallei, formerly known as Pseudomonas pseudomallei, is a gram-negative, aerobic, bipolar, motile, rod-shaped bacterium. B. pseudomallei infections (melioidosis) can be severe and even fatal in both humans and animals. This environmental saprophyte is endemic to Southeast Asia and northern Australia, but it has also been found in other tropical and subtropical areas of the world.^7,22,32,42 The bacterium is usually found in soil and water in endemic areas and is transmitted to humans and animals primarily through percutaneous inoculation, ingestion, or inhalation of a contaminated source.^{8, 22,28,32,42} Human-to-human, animal-to-animal, and animal-to-human spread are rare.^8,32 In December 2012, the National Select Agent Registry designated B. pseudomallei as a Tier 1 overlap select agent.³⁹ Organisms classified as Tier 1 agents present the highest risk of deliberate misuse, with the most significant potential for mass casualties or devastating effects to the economy, critical infrastructure, or public confidence. Select agents with this status have the potential to pose a severe threat to human and animal health or safety or the ability to be used as a biologic weapon.³⁹Melioidosis in humans can be challenging to diagnose and treat because the organism can remain latent for years and is resistant to many antibiotics.^12,37,41 B. pseudomallei can survive in phagocytic cells, a phenomenon that may be associated with latent infections.^19,38 The incubation period in naturally infected animals ranges from 1 d to many years, but symptoms typically appear 2 to 4 wk after exposure.^13,17,35,38 Disease generally presents in 1 of 2 forms: localized infection or septicemia.²² Multiple methods are used to diagnose melioidosis, including immunofluorescence, serology, and PCR analysis, but isolation of the bacteria from blood, urine, sputum, throat swabs, abscesses, skin, or tissue lesions remains the ‘gold standard.’^9,22,40,42 The prognosis varies based on presentation, time to diagnosis, initiation of appropriate antimicrobial treatment, and underlying comorbidities.^7,28,42 Currently, there is no licensed vaccine to prevent melioidosis.There are several published reports of naturally occurring melioidosis in a variety of nonhuman primates (NHP; 2,10,13,17,25,30,31,35 The first reported case of melioidosis in monkeys was recorded in 1932, and the first published case in a macaque species was in 1966.³⁰ In the United States, there have only been 7 documented cases of NHP with B. pseudomallei infection.^2,13,17 All of these cases occurred prior to the classification of B. pseudomallei as a select agent. Clinical signs in NHP range from subclinical or subacute illness to acute septicemia, localized infection, and chronic infection. NHP with melioidosis can be asymptomatic or exhibit clinical signs such as anorexia, wasting, purulent drainage, subcutaneous abscesses, and other soft tissue lesions. Lymphadenitis, lameness, osteomyelitis, paralysis and other CNS signs have also been reported.^{2,7,10,22,28,32} In comparison, human''s clinical signs range from abscesses, skin ulceration, fever, headache, joint pain, and muscle tenderness to abdominal pain, anorexia, respiratory distress, seizures, and septicemia.^7,9,21,22

Table 1.

Summary of reported cases of naturally occurring Burkholderia pseudomalleiinfections in nonhuman primates

Tomato BRI1 and systemin wound signalling

Brassinosteroids (BRs) are perceived by Brassinosteroid Insensitive 1 (BRI1), that encodes a leucine-rich repeat receptor kinase. Tomato BRI1 has previously been implicated in both systemin and BR signalling. The role of tomato BRI1 in BR signalling was confirmed, however it was found not to be essential for systemin/wound signalling. Tomato roots were shown to respond to systemin but this response varied according to the species and growth conditions. Overall the data indicates that mutants defective in tomato BRI1 are not defective in systemin-induced wound signalling and that systemin perception can occur via a non-BRI1 mechanism.Key words: tomato BRI1, brassinosteroids, systemin, wound signallingBrassinosteroids (BRs) are steroid hormones that are essential for normal plant growth. The most important BR receptor in Arabidopsis is BRASSINOSTERIOD INSENSITIVE 1 (BRI1), a serine/threonine kinase with a predicted extracellular domain of ∼24 leucine-rich repeats (LRRs).^1,2 BRs bind to BRI1 via a steroid-binding domain that includes LRR 21 and a so-called “island” domain.^2,3 In tomato a BRI1 orthologue has been identified that when mutated, as in the curl3 (cu3) mutation, results in BR-insensitive dwarf plants.⁴ Tomato BRI1 has also been purified as a systemin-binding protein.⁵ Systemin is an eighteen amino acid peptide, which is produced by post-translational cleavage of prosystemin. Systemin has been implicated in wound signalling and is able to induce the production of jasmonate, protease inhibitors (PIN) and rapid alkalinization of cell suspensions (reviewed in ref. ⁶).To clarify whether tomato BRI1 was indeed a dual receptor it was important to first confirm its role in BR signalling. Initially this was carried out by genetic complementation of the cu3 mutant phenotype.⁷ Overexpression of tomato BRI1 restored the dwarf phenotype and BR sensitivity and normalized BR levels (

De novo mammalian prion synthesis

Prions are responsible for a heterogeneous group of fatal neurodegenerative diseases. They can be sporadic, genetic, or infectious disorders involving post-translational modifications of the cellular prion protein (PrP^C). Prions (PrP^Sc) are characterized by their infectious property and intrinsic ability to convert the physiological PrP^C into the pathological form, acting as a template. The “protein-only” hypothesis, postulated by Stanley B. Prusiner, implies the possibility to generate de novo prions in vivo and in vitro. Here we describe major milestones towards proving this hypothesis, taking into account physiological environment/s, biochemical properties and interactors of the PrP^C.Key words: prion protein (PrP), prions, amyloid, recombinant prion protein, transgenic mouse, protein misfolding cyclic amplification (PMCA), synthethic prionPrions are responsible for a heterogeneous group of fatal neurodegenerative diseases (1 They can be sporadic, genetic or infectious disorders involving post-translational modifications of the cellular prion protein (PrP^C).² Prions are characterized by their infectious properties and by their intrinsic ability to encipher distinct biochemical properties through their secondary, tertiary and quaternary protein structures. In particular, the transmission of the disease is due to the ability of a prion to convert the physiological PrP^C into the pathological form (PrP^Sc), acting as a template.³ The two isoforms of PrP appear to be different in terms of protein structures, as revealed by optical spectroscopy experiments such as Fourier-transform infrared and circular dichroism.⁴ PrP^C contains 40% α-helix and 3% β-sheet, while the pathological isoform, PrP^Sc, presents approximately 30% α-helix and 45% β-sheet.^4,5 PrP^Sc differs from PrP^C because of its altered physical-chemical properties such as insolubility in non-denaturing detergents and proteinases resistance.^2,6,7

Table 1

The prion diseases

Genome-wide analysis of lipoxygenase gene family in Arabidopsis and rice

The enzymes called lipoxygenases (LOXs) can dioxygenate unsaturated fatty acids, which leads to lipoperoxidation of biological membranes. This process causes synthesis of signaling molecules and also leads to changes in cellular metabolism. LOXs are known to be involved in apoptotic (programmed cell death) pathway, and biotic and abiotic stress responses in plants. Here, the members of LOX gene family in Arabidopsis and rice are identified. The Arabidopsis and rice genomes encode 6 and 14 LOX proteins, respectively, and interestingly, with more LOX genes in rice. The rice LOXs are validated based on protein alignment studies. This is the first report wherein LOXs are identified in rice which may allow better understanding the initiation, progression and effects of apoptosis, and responses to bitoic and abiotic stresses and signaling cascades in plants.Key words: apoptosis, biotic and abiotic stresses, genomics, jasmonic acid, lipidsLipoxygenases (linoleate:oxygen oxidoreductase, EC 1.13.11.-; LOXs) catalyze the conversion of polyunsaturated fatty acids (lipids) into conjugated hydroperoxides. This process is called hydroperoxidation of lipids. LOXs are monomeric, non-heme and non-sulfur, but iron-containing dioxygenases widely expressed in fungi, animal and plant cells, and are known to be absent in prokaryotes. However, a recent finding suggests the existence of LOX-related genomic sequences in bacteria but not in archaea.¹ The inflammatory conditions in mammals like bronchial asthama, psoriasis and arthritis are a result of LOXs reactions.² Further, several clinical conditions like HIV-1 infection,³ disease of kidneys due to the activation of 5-lipoxygenase,^4,5 aging of the brain due to neuronal 5-lipoxygenase⁶ and atherosclerosis⁷ are mediated by LOXs. In plants, LOXs are involved in response to biotic and abiotic stresses.⁸ They are involved in germination⁹ and also in traumatin and jasmonic acid biochemical pathways.^10,11 Studies on LOX in rice are conducted to develop novel strategies against insect pests¹² in response to wounding and insect attack,¹³ and on rice bran extracts as functional foods and dietary supplements for control of inflammation and joint health.¹⁴ In Arabidopsis, LOXs are studied in response to natural and stress-induced senescence,¹⁵ transition to flowering,¹⁶ regulation of lateral root development and defense response.¹⁷The arachidonic, linoleic and linolenic acids can act as substrates for different LOX isozymes. A hydroperoxy group is added at carbons 5, 12 or 15, when arachidonic acid is the substrate, and so the LOXs are designated as 5-, 12- or 15-lipoxygenases. Sequences are available in the database for plant lipoxygenases (EC:1.13.11.12), mammalian arachidonate 5-lipoxygenase (EC:1.13.11.34), mammalian arachidonate 12-lipoxygenase (EC:1.13.11.31) and mammalian erythroid cell-specific 15-lipoxygenase (EC:1.13.11.33). The prototype member for LOX family, LOX-1 of Glycine max L. (soybean) is a 15-lipoxygenase. The LOX isoforms of soybean (LOX-1, LOX-2, LOX-3a and LOX-3b) are the most characterized of plant LOXs.¹⁸ In addition, five vegetative LOXs (VLX-A, -B, -C, -D, -E) are detected in soybean leaves.¹⁹ The 3-dimensional structure of soybean LOX-1 has been determined.^20,21 LOX-1 was shown to be made of two domains, the N-terminal domain-I which forms a β-barrel of 146 residues, and a C-terminal domain-II of bundle of helices of 693 residues²¹ (Fig. 1). The iron atom was shown to be at the centre of domain-II bound by four coordinating ligands, of which three are histidine residues.²²Open in a separate windowFigure 1Three-dimensional structure of soybean lipoxygenase L-1. The domain I (N-terminal) and domain II (C-terminal) are indicated. The catalytic iron atom is embedded in domain II (PDB ID-1YGE).²¹This article describes identification of LOX genes in Arabidopsis and rice. The Arabidopsis genome encodes for six LOX proteins²³ (www.arabidopsis.org) (LocusAnnotationNomenclatureA^*B^*C^*AT1G55020lipoxygenase 1 (LOX1)LOX185998044.45.2049AT1G17420lipoxygenase 3 (LOX3)LOX3919103725.18.0117AT1G67560lipoxygenase family proteinLOX4917104514.68.0035AT1G72520lipoxygenase, putativeLOX6926104813.17.5213AT3G22400lipoxygenase 5 (LOX5)LOX5886101058.86.6033AT3G45140lipoxygenase 2 (LOX2)LOX2896102044.75.3177Open in a separate window^*A, amino acids; B, molecular weight; C, isoelectric point.Interestingly, the rice genome (rice.plantbiology.msu.edu) encodes for 14 LOX proteins as compared to six in Arabidopsis (​and22). Of these, majority of them are composed of ∼790–950 aa with the exception for loci, LOC_Os06g04420 (126 aa), LOC_Os02g19790 (297 aa) and LOC_Os12g37320 (359 aa) (Fig. 2).Open in a separate windowFigure 2Protein alignment of rice LOXs and vegetative lipoxygenase, VLX-B,²⁸ a soybean LOX (AA B67732). The 14 rice LOCs are indicated on left and sequence position on right. Gaps are included to improve alignment accuracy. Figure was generated using ClustalX program.

Table 2

Genes encoding lipoxygenases in rice

Indirect effects of tending ants on holm oak volatiles and acorn quality

The indirect effect of ants on plants through their mutualism with honeydew-producing insects has been extensively investigated. Honeydew-producing insects that are tended by ants impose a cost on plant fitness and health by reducing seed production and/or plant growth. This cost is associated with sap intake and virus transmissions but may be overcompesated by tending ants if they deter or prey on hebivorous insects. The balance between cost and benefits depends on the tending ant species. In this study we report other indirect effects on plants of the mutualism between aphids and ants. We have found that two Lasius ant species, one native and the other invasive, may change the composition of volatile organic compounds (VOCs) of the holm oak (Quercus ilex) blend when they tend the aphid Lachnus roboris. The aphid regulation of its feeding and honeydew production according to the ant demands was proposed as a plausible mechanism that triggers changes in VOCs. Additionally, we now report here that aphid feeding, which is located most of the time on acorns cap or petiole, significantly increased the relative content of linolenic acid in acorns from holm oak colonized by the invasive ant. This acid is involved in the response of plants to insect herbivory as a precursor or jasmonic acid. No effect was found on acorn production, germination or seedlings quality. These results suggest that tending-ants may trigger the physiological response of holm oaks involved in plant resistance toward aphid herbivory and this response is ant species-dependent.Key words: tended aphid, invasive ants, linolenic acid, jasmonic acid, monoterpene emissionsTo achieve an indirect effect it is necessary to have a minimun of three species, two focal species that interact directly and an associate species whose presence promotes an indirect effect on one or both focal species. In general, indirect effects of a third species are defined by how and to what degree a pairwise species interaction is influenced by the presence and density of this third species.¹ There are several examples of interactions presenting indirect effects: apparent competition,¹ facilitation,² tri-trophic level interactions,³ cascading effects⁴ and exploitative competition. 5 But, indirect effects have been studied most extensively in the context of trophic cascades when top predators are removed⁶ or added⁷ and in the context of mutualisms.^8–10 Usually, indirect effects are investigated as changes in abundance of the focal species occur. However, indirect effects may result in biologically significant changes in a species that are not reflected only to its abundance.¹¹ There are many examples of changes in physiology, behavior, morphology and/or genotypic composition of the focal species.^11,12 These changes on density and/or morphological, physiological and behavioral traits of the focal species are not mutually exclusive, and all can act at the same time.¹³ The magnitude and direction of both direct and indirect effects should influence the relative resilience of communities to perturbation, which in turn will affect species coexistence and community evolution.¹⁴ In this regard, indirect effects had been postulated as one of the main forces structuring communities² and shaping the evolution of communities.¹⁴In terrestrial communities ants interact with plants both directly and indirectly. They can disperse or consume seeds, feed from specialized plant structures such as food bodies and extrafloral nectaries, act as or deter pollinitators, prey on herbivorous insects and/or develop mutualisms with honeydew-producing insects indirectly modifying plant fitness.^15–17 Additionally, through their nesting activities in soil, ants increase soil nutrient content available to plants, may change water infiltration and soil holding-capacity and modify biodiversity and abundance of soil organisms related to the decomposition process.^18,19 As a consequence of their activities, ants may thus change behavior, density, physiology or fitness of other species.^12,22,23 In the case of ants that tend honeydew-producing insects, evidence shows that their attention may change some traits of insect life history, 22 their abundance or physiology.¹⁸ For the plant, the net outcome of the mutualism between ants and honeydew-producing insects will depend on the balance between the costs for plant fitness via consumption of plant sap and transmission of plant pathogens and the benefit of ants deterring herbivorous insects.^18,23 As a consequence, plant seed production, pod production or even plant growth may decrease when the cost of honeydew-producing insects exceed the benefit provided by tending ants.^18,23Recently, we have described the changes that two tending ant species may exert indirectly on monoterpene emissions of holm oak (Quercus ilex) saplings through its mutualism with Lachnus roboris aphids.²⁴ One of these tending ant species was Lasius neglectus, an invasive ant species that displaces the local ant Lasius grandis. We found that aphids feeding on holm oak increased the emission of total volatile organic carbon (VOCs) by 31%. In particular, aphids feeding elicited the emission of a new monoterpene, Δ³-carene, and increased the emission of myrcene (mean ± SE; sapling alone: 0.105 ± 0.011 µg g⁻¹ h⁻¹; sapling plus not tended aphid: 0.443 ± 0.057 µg g⁻1 h⁻1) and γ-terpinene (sapling alone: 0.0013 ± 0.0001; sapling plus not tended aphid: 0.0122 ± 0.0022 µg g⁻1 h⁻1) (Mann-Whitney, sapling alone vs. sapling plus not tended aphids, U_4,4 = 0, p < 0.05 for both compounds). Changes of VOC emission in response to aphid infestation were noticed also in boreal trees.²⁴ When the aphids became tended by the invasive ant, L. neglectus, VOCs emissions increased only 19% because myrcene, the main compound of the blend, decreased significantly (25 When our data was recalculated on leaf area basis (nmol m⁻² s⁻¹), the general pattern was the same independently of the units, but the differences among treatments were not statistically significant (26 These slight differences in the statitiscal significance of the differences of VOC emissions depending on the reference unit may be due to differences in leaf morphology, i.e., changes of leaf area and mass. However, in our study, all holm oaks showed a similar leaf morphology among treatments (Kruskal-Wallis, leaf mass: H_3,20 = 2.16, p = 0.53; leaf area: H_3,20 = 2.64, p = 0.45) (24^,27 This lack of consistence of aphid effect on leaf area and mass limits the development of a clear pattern linking aphids feeding, leaf area or mass and VOC emissions. On the other hand, to achieve statistical significance of emitted VOCs among treatments, values should differ strongly given the high variability of VOC emission within treatments.²⁶ Under this scenario, we recommend giving the values of leaf morphology and to give VOC emissions on both unit bases to facilite comparisons among different studies.

Table 1

Means and standard error of the emission rates of the main compounds emitted by Quercus ilex saplings (n = 4 for T1 and T2 and n = 8 for T3) infested with untended aphids (T1) or infested with aphids tended by the native ant Lasius grandis (T2) or by the invasive ant Lasius neglectus (T3)

Sources of floral scent variation: Can environment define floral scent phenotype?

Studies of floral scent generally assume that genetic adaptation due to pollinator-mediated natural selection explains a significant amount of phenotypic variance, ignoring the potential for phenotypic plasticity in this trait. In this paper, we assess this latter possibility, looking first at previous studies of floral scent variation in relation to abiotic environmental factors. We then present data from our own research that suggests among-population floral scent variation is determined, in part, by environmental conditions and thus displays phenotypic plasticity. Such an outcome has strong ramifications for the study of floral scent variation; we conclude by presenting some fundamental questions that should lead to greater insight into our understanding of the evolution of this trait, which is important to plant-animal interactions.Key words: abiotic factors, aromatics, floral scent, GxE interaction, phenotypic plasticity, pollination, terpenoids, volatilesFloral scent is thought to function as a major non-visual attractive cue for many pollinators in a large number of plant systems^1,2 and therefore most research on this plant trait has proceeded in the context of pollination ecology. Such studies have revealed the physiological and behavioral responses of pollinators to various floral volatiles (reviewed in refs. ³ and ⁴), convergent evolution of odor phenotypes attractive to specific pollinator classes (reviewed in refs. ⁵ and ⁶), reproductive isolation of plant species due to differences in pollinator attraction by scent,⁷ and instances of deception in which flowers mimic insect pheromones to effect pollination.⁸ Together, this body of evidence suggests that specific floral scent profiles can have important implications for the reproductive potential of many plant species.This pollinator-centered viewpoint has carried through to research on floral scent variation, including our most recent work on the insect-pollinated species Hesperis matronalis (Brassicaceae).⁹ Such studies usually suggest that the floral scent variation commonly found within and among individuals, populations and species (reviewed in ref. ²) is due to genetic differentiation as a result of selection by pollinators over time (reviewed in ref. ¹⁰). But an organism''s genes are only one factor determining phenotype. Both biotic (living) and abiotic (non-living) environmental conditions can profoundly affect phenotype expression, leading to significant variation. For plants, abiotic factors such as climate and soil chemistry can have particularly strong effects on phenotypes. When these environmental conditions cause changes in phenotype, we would say that a trait displays phenotypic plasticity.^11–13 A number of studies have uncovered phenotypic plasticity for many different plant traits.¹² However, while phenotypic variation in floral scent has been well-documented^1,2 and correlated with variation in biotic factors like pollinator behavior,^14–17 these studies were decidedly focused on natural selection, rather than phenotypic plasticity, as an organizational framework.However, in examining the scientific literature on floral scent, we found four studies in which the effects of naturally variable abiotic factors on floral scent profiles were examined, three of which were performed by the same research group (18^–21 (21). Moreover, these studies are decidedly not analyzed and interpreted using standard protocols for phenotypic plasticity studies.¹³

Table 1

A survey of previous studies examining changes in floral scent phenotype due to abiotic factors

Immunomodulation by Mesenchymal Stem Cells in Veterinary Species

Mesenchymal stem cells (MSC) are adult-derived multipotent stem cells that have been derived from almost every tissue. They are classically defined as spindle-shaped, plastic-adherent cells capable of adipogenic, chondrogenic, and osteogenic differentiation. This capacity for trilineage differentiation has been the foundation for research into the use of MSC to regenerate damaged tissues. Recent studies have shown that MSC interact with cells of the immune system and modulate their function. Although many of the details underlying the mechanisms by which MSC modulate the immune system have been defined for human and rodent (mouse and rat) MSC, much less is known about MSC from other veterinary species. This knowledge gap is particularly important because the clinical use of MSC in veterinary medicine is increasing and far exceeds the use of MSC in human medicine. It is crucial to determine how MSC modulate the immune system for each animal species as well as for MSC derived from any given tissue source. A comparative approach provides a unique translational opportunity to bring novel cell-based therapies to the veterinary market as well as enhance the utility of animal models for human disorders. The current review covers what is currently known about MSC and their immunomodulatory functions in veterinary species, excluding laboratory rodents.Abbreviations: AT, adipose tissue; BM, Bone marrow; CB, umbilical cord blood; CT, umbilical cord tissue; DC, dendritic cell; IDO, indoleamine 2;3-dioxygenase; MSC, mesenchymal stem cells; PGE₂, prostaglandin E2; VEGF, vascular endothelial growth factorMesenchymal stem cells (MSC, alternatively known as mesenchymal stromal cells) were first reported in the literature in 1968.³⁹ MSC are thought to be of pericyte origin (cells that line the vasculature)^21,22 and typically are isolated from highly vascular tissues. In humans and mice, MSC have been isolated from fat, placental tissues (placenta, Wharton jelly, umbilical cord, umbilical cord blood), hair follicles, tendon, synovial membrane, periodontal ligament, and every major organ (brain, spleen, liver, kidney, lung, bone marrow, muscle, thymus, pancreas, skin).^23,121 For most current clinical applications, MSC are isolated from adipose tissue (AT), bone marrow (BM), umbilical cord blood (CB), and umbilical cord tissue (CT; 11,87,99 Clinical trials in human medicine focus on the use of MSC both for their antiinflammatory properties (graft-versus-host disease, irritable bowel syndrome) and their ability to aid in tissue and bone regeneration in combination with growth factors and bone scaffolds (clinicaltrials.gov).¹³¹ For tissue regeneration, the abilities of MSC to differentiate and to secrete mediators and interact with cells of the immune system likely contribute to tissue healing (Figure 1). The current review will not address the specific use of MSC for orthopedic applications and tissue regeneration, although the topic is covered widely in current literature for both human and veterinary medicine.^57,62,90

Table 1.

Tissues from which MSC have been isolated

Induction of systemic resistance in rice by leaf extracts of Zizyphus jujuba and Ipomoea carnea against Rhizoctonia solani

Plants accumulate a great diversity of natural products, many of which confer protective effects against phytopathogenic attack. Earlier we had demonstrated that the leaf extracts of Zizyphus jujuba and Ipomoea carnea inhibit the in vitro mycelial growth of Rhizoctonia solani, and effectively reduce the incidence of sheath blight disease in rice.⁷ Here we demonstrate that foliar application of the aqueous leaf extracts of Z. jujuba and I. carnea followed by challenge inoculation with R. solani induces systemic resistance in rice as evident from significantly increased accumulation of pathogenesis-related proteins such as chitinase, β-1,3-glucanase and peroxidase, as well as defense-related compounds such as phenylalanine ammonia-lyase and phenolic substances. Thin layer chromatographic separation of secondary metabolites revealed presence of alkaloid and terpenoid compounds in the leaf extracts of Z. jujuba that exhibited toxicity against R. solani under in vitro condition. Thus, the enhanced sheath blight resistance in rice seedlings treated with leaf extracts of Z. jujuba or I. carnea can be attributed to the direct inhibitory effects of these leaf extracts as well as their ability to elicit systemic resistance against R. solani.Key words: sheath blight, Zizyphus jujuba, Ipomoea carnea, Rhizoctonia solani, induced systemic resistance, antimicrobial compoundsSheath blight disease of rice, caused by Rhizoctonia solani, has become a major production constraint in intensive rice cropping systems where semi-dwarf, nitrogen-responsive and high-yielding rice cultivars are grown. The disease causes an annual yield loss of upto 50%.¹ R. solani is both soil- and water-borne, and can infect more than 27 families of both monocot and dicot species.² Natural host genetic resistance to R. solani has not been recorded in cultivars or wild relatives of rice.³ Several broad spectrum fungicides have been recommended for control of sheath blight, however, chemical method of disease management is neither practical due to high cost of fungicides nor sustainable as it can affect the balance of ecosystem by destroying beneficial microbial population. In addition, the environmental pollution problems associated with indiscriminate use of synthetic pesticides have prompted investigations on exploiting bio-pesticides of plant and microbial origin.Plants accumulate an enormous variety of over 100,000 secondary metabolites,⁴ which can act as pre-existing chemical inhibitors to invading pathogens and/or help strengthen defense response of host plant. The pre-formed infectional barriers in plants are generally referred to as “phytoanticipins;” whereas, the antimicrobial compounds that are synthesized de novo in response to pathogen attack are referred to as “phytoalexins.”⁵ Because of years of selective breeding leading to removal of natural products, the endogenous levels of phytoanticipins in commonly cultivated crop species are generally low and often not sufficient to fight pathogen attack, effectively.⁴ Various weed species and wild relatives of crop plants that are not subjected to selective breeding are believed to contain higher levels of antimicrobial compounds, consistent with their ability to fight invading pathogens more effectively than cultivated crop species. Identification of such weed/plant species that are enriched with antimicrobial principles, isolation of bio-active compounds from them, and application in the form of concentrated formulations to crop plants can augment their disease resistance capability by directly inhibiting the growth of pathogen and inducing defense responses. Indeed, the antimicrobial properties of tissue extracts of several weed/plant species have been reported by a number of research groups world-wide, especially in Asia and Latin America.^6–13Earlier, we had evaluated the antimicrobial activity of leaf extracts of 16 different plant species belonging to 16 different families and demonstrated that leaf extracts of most of these plant species exhibit growth-inhibitory activities against R. solani and Xanathomoas oryzae pv. oryzae (Xoo).⁷ Among these, the leaf extracts of Datura metel were found to be the most effective in inhibiting the mycelial growth and sclerotia formation of R. solani, and the growth of Xoo, as well as in reducing the incidence of sheath blight and bacterial blight diseases caused by these pathogens, respectively, under greenhouse condition.⁷ We further demonstrated that rice seedlings treated with leaf extracts of D. metel accumulated significantly higher levels of pathogenesis-related (PR) proteins and other defense related compounds following challenge inoculation with R. solani or Xoo.⁷ Our attempts to identify biologically active compounds from D. metel revealed the presence of a withanolide compound “daturilin” that exhibited remarkable antibacterial activity against Xoo.⁷Apart from D. metel, two other plants species, Zizyphus jujuba and Ipomoea carnea, were found to possess remarkable antifungal activity against R. solani.⁷ Z. jujuba is a thorny rhamnaceous plant that is widely distributed in Europe and South-eastern Asia. I. carnea of convolvulaceae family, commonly known as morning glory, is a toxic weed found in abundance in India, Brazil, the United States and other countries.¹⁴ Both of these plant species have allelopathic effect and are commonly used in folklore medicine for curing multiple diseases.^15–18 The aqueous and methanol leaf extracts of Z. jujuba and I. carnea have been found to be highly effective in reducing in vitro mycelial growth, and therefore, sclerotia production of R. solani.⁷ In the greenhouse experiments, rice seedlings sprayed with leaf extracts of Z. jujuba and I. carnea exhibited 44 and 34% reduction in severity of sheath blight disease over the control, respectively.⁷ While these findings are encouraging, the mechanisms by which the leaf extracts of Z. jujuba and I. carnea modulate defense responses in rice have not yet been explored.Plants are endowed with defense genes which remain quiescent or are expressed at basal levels in healthy plants. Activation of defense genes results in induction of systemic resistance in host plant; this defense response, designated as induced systemic resistance (ISR), plays an important role in development of disease resistance.¹⁹ The onset of ISR in plants correlates with accumulation of phytoalexins and increased activity of PR proteins such as chitinases, β-1,3-glucanases and peroxidases;^20–23 consequently, PR proteins are generally used as ISR markers.¹⁹ The classical inducers of ISR include both biotic and abiotic factors, including disease causing microorganisms themselves,^24,25 plant growth promoting rhizobacteria,^22,26 chemicals^27,28 and natural plant products.^{7,10,12,13,29,30} Plant products have been considered as one of the major groups of compounds that induce ISR. To date, extracts of at least a few plant species have been reported to contain allelopathic substances which can act as elicitors and induce systemic resistance in host plants resulting in reduction or inhibition of disease development.^7,10,12,13In the present study, with the objective of understanding the mechanisms of disease suppression by leaf extracts of Z. jujuba and I. carnea, we investigated their ability to induce ISR in rice by analyzing the activities of ISR markers including PR-proteins and other defense enzymes involved in phenylpropanoid metabolism. The changes in activities of chitinase, β-1,3-glucanase, peroxidase, phenylalanine ammonia-lyase (PAL) and phenolic compounds induced in rice seedlings that were elicited with leaf extracts (at 1:10 dilution; w/v) of Z. jujuba or I. carnea and infected with R. solani were analyzed, and compared to changes in non-elicited and uninfected seedlings. Rice seedlings that were both elicited with leaf extracts of Z. jujuba or I. carnea and infected with R. solani accumulated significantly higher levels (2–5-fold) of ISR markers as compared to non-elicited and/or uninfected seedlings (Fig. 1). About two-fold increase in activities of ISR markers was also observed in seedlings that were either infected but not elicited or elicited but not infected; however, this increase was significantly lower than the changes in seedlings that were both elicited and infected (Fig. 1). Although the activity of all ISR markers began to increase around or after 24 h post-infection, at least two distinct induction patterns were observed. For instance, the activities of chitinase and phenolic substances gradually increased to reach maximum levels at 164 h post-infection (Fig. 1A and E); whereas, the activities of β-1,3-glucanase, peroxidase and PAL reached maximum levels at 72 to 96 h post-infection and decreased thereafter (Fig. 1B–D). The leaf extracts of Z. jujuba were found slightly more effective in inducing ISR markers than the leaf extracts of I. carnea. There was no significant change in the activity of ISR markers in control seedlings sprayed with sterile distilled water (Fig. 1). Collectively, these results suggested that the leaf extracts of Z. jujuba and I. carnea have the ability to induce systemic resistance in rice seedlings infected with R. solani. The fungitoxicity of the leaf extracts of Z. jujuba and I. carnea ⁷ combined with their ability to elicit ISR is possibly responsible for low sheath blight disease incidence observed in rice seedlings treated with these leaf extracts.⁷Open in a separate windowFigure 1Activity of ISR markers and defense-related compounds in rice seedlings elicited with the leaf extracts of Zizyphus jujuba or Ipomoea carnea and challenge inoculated with Rhizoctonia solani. Total activity of chitinase (A), β-1,3-glucanase (B), peroxidase (C) phenylalanine ammonia-lyase (PAL; D) and phenolic substances (E) was analyzed in rice seedlings. The inoculation of rice seedlings with R. solani was performed 45 days after planting. Spraying of leaf extracts (1:10 dilution; w/v) of Z. jujuba or I. carnea was performed two days prior to inoculation. Tissue samples (sheath) from elicited and/or infected seedlings were collected for analysis at various time intervals.The in vitro antimicrobial and in vivo disease inhibitory effects of natural plant products are generally attributed to the allelopathic substances present in them. However, very few attempts have been made to purify and characterize active principles from bio-active natural plant products. We have previously identified a withanolide compound from leaf extracts of D. metel which exhibited antibacterial activity against Xoo.⁷ Both Z. jujuba and I. carnea are rich source of secondary metabolites including alkaloids, terpenoids, flavonoids and phenolic compounds.^31–35 To determine the composition of bio-active ingredients within the leaf extracts of Z. jujuba and I. carnea, we performed thin layer chromatographic separation of alkaloid, terpenoid and phenolic compounds. The partially purified compounds, as reported in Leaf extractR_f valueAnti-fungal activity against R. solani^*VisibleIodine vaporsUV-lightSpray reagentPhenolic substances¹Z. jujuba0.6960.696-0.696-I. carnea-0.807-0.807-Terpenoid compounds²Z. jujuba---0.189-0.3580.3580.3580.3585.1 mm---0.4463.7 mmI. carnea-0.5900.5900.590-Alkaloid compounds³Z. jujuba-0.784-0.7845.1 mmI. carnea-0.806-0.806-Open in a separate window^*Inhibition zone diameter (mm) as mean of triplicate tests.¹Solvent-acetic acid:chloroform (1:9); Spray reagent-Diazotised sulphanilic acid.²Solvent-methanol:chloroform (2:9); Spray reagent-10% vanillin-sulphuric acid.³Solvent-methanol:chloroform (1:1); Spray reagent-Drag endorffs reagent.In conclusion, our results together with several other reports in the literature have established that natural plant products possess antimicrobial substances that can inhibit the growth of the pathogens and augment disease resistance capability of plants by eliciting ISR in host plants. In the immediate future, identification and characterization of additional novel bio-active compounds from natural plant products is essential for developing commercial formulations of potential use in controlling pathogenic diseases in crop plants.Rice cultivar, IR-50 (susceptible to sheath blight) and virulent isolate of R. solani (RS7 Anastamosis group AG1),³⁶ were used in all experiments. The leaf tissues of Z. jujuba and I. carnea were collected from local areas around Coimbatore, India and aqueous extracts were prepared, as described previously in reference ⁷. Forty-five-day-old rice seedlings were sprayed with either aqueous leaf extracts (1:10 dilution) or sterile distilled water, two-days prior to inoculation with sclerotia of R. solani.³⁷ Sheath tissues from infected seedlings were collected at various time intervals, including 0, 24, 48, 72, 96 and 164 h after pathogen inoculation. The changes in the chitinase and peroxidase activities were determined by colorimetric assays, as described previously by Boller and Mauch,³⁸ and Hammerschmidt et al.³⁹ respectively. β-1,3-glucanase activity was assayed by the laminarin-dinitrosalicylic acid method.⁴⁰ PAL activity was determined as the rate of conversion of L-phenylalanine to trans-cinnamic acid at 290 nm as described by Dickerson et al.⁴¹ The amount of trans-cinnamic acid synthesized was calculated using its extinction coefficient of 9,630 M⁻¹. Estimation of phenolic substances was carried out as described previously in reference ⁷.TLC was carried out on 20 × 20 cm glass plate coated with0.5 mm thickness silica gel. Twenty microliters of Z. jujuba and I. carnea leaf extracts (1 g/ml) were spotted on each plate. The mixture of solvents comprising acetic acid:chloroform (1:9), methanol:chloroform (2:9) or methanol:chloroform (1:1) were used to develop the chromatograms for detection of phenolic, terpenoid or alkaloid compounds, respectively. The developed chromatograms were observed under visible, UV light and after exposing to iodine vapours. Additionally, the chemical class specific visualization spray reagents were used for detection of phenolic substances (Diazotized sulphanilic acid), terpenoids (10% vanillin-sulphuric acid) and alkaloids (Dragendorffs reagent). Preparative TLC was carried out using 2 mm thickness silica gel. The Rf value of each spot detected on the chromatogram was recorded. The silica gel corresponding to each spot was scraped off and the chemical compound was eluted using sterile water. The eluted compound was tested for its antimicrobial activity using the inhibition zone technique.⁴²     

Prion interference with multiple prion isolates

Co-inoculation of prion strains into the same host can result in interference, where replication of one strain hinders the ability of another strain to cause disease. The drowsy (DY) strain of hamster-adapted transmissible mink encephalopathy (TME) extends the incubation period or completely blocks the hyper (HY) strain of TME following intracerebral, intraperitoneal or sciatic nerve routes of inoculation. However, it is not known if the interfering effect of the DY TME agent is exclusive to the HY TME agent by these experimental routes of infection. To address this issue, we show that the DY TME agent can block hamster-adapted chronic wasting disease (HaCWD) and the 263K scrapie agent from causing disease following sciatic nerve inoculation. Additionally, per os inoculation of DY TME agent slightly extends the incubation period of per os superinfected HY TME agent. These studies suggest that prion strain interference can occur by a natural route of infection and may be a more generalized phenomenon of prion strains.Key words: prion diseases, prion interference, prion strainsPrion diseases are fatal neurodegenerative diseases that are caused by an abnormal isoform of the prion protein, PrP^Sc.¹ Prion strains are hypothesized to be encoded by strain-specific conformations of PrP^Sc resulting in strain-specific differences in clinical signs, incubation periods and neuropathology.^2–7 However, a universally agreed upon definition of prion strains does not exist. Interspecies transmission and adaptation of prions to a new host species leads to the emergence of a dominant prion strain, which can be due to selection of strains from a mixture present in the inoculum, or produced upon interspecies transmission.^8,9 Prion strains, when present in the same host, can interfere with each other.Prion interference was first described in mice where a long incubation period strain 22C extended the incubation period of a short incubation period strain 22A following intracerebral inoculation.¹⁰ Interference between other prion strains has been described in mice and hamsters using rodent-adapted strains of scrapie, TME, Creutzfeldt-Jacob disease and Gerstmannn-Sträussler-Scheinker syndrome following intracerebral, intraperitoneal, intravenous and sciatic nerve routes of inoculation.^10–15 We previously demonstrated the detection of PrP^Sc from the long incubation period DY TME agent correlated with its ability to extend the incubation period or completely block the superinfecting short incubation period HY TME agent from causing disease and results in a reduction of HY PrP^Sc levels following sciatic nerve inoculation.¹² However, it is not known if a single long incubation period agent (e.g., DY TME) can interfere with more than one short incubation period agent or if interference can occur by a natural route of infection.To examine the question if one long incubation period agent can extend the incubation period of additional short incubation period agents, hamsters were first inoculated in the sciatic nerve with the DY TME agent 120 days prior to superinfection with the short-incubation period agents HY TME, 263K scrapie and HaCWD.^16–18 The HY TME and 263K scrapie agents have been biologically cloned and have distinct PrP^Sc properties.^19,20 The HaCWD agent used in this study is seventh hamster passage that has not been biologically cloned and therefore will be referred to as a prion isolate. Sciatic nerve inoculations were performed as previously described.^11,12 Briefly, hamsters were inoculated with 103.0 i.c. LD₅₀ of the DY TME agent or equal volume (2 µl of a 1% w/v brain homogenate) of uninfected brain homogenate 120 days prior to superinfection of the same sciatic nerve with either 10^4.6 i.c. LD₅₀ of the HY TME agent, 10^5.2 i.c. LD₅₀ of the HaCWD agent or 10^4.6 i.c. LD₅₀/g 263K scrapie agent (Bartz J, unpublished data).^16,18,21 Animals were observed three times per week for the onset of clinical signs of HY TME, 263K and HaCWD based on the presence of ataxia and hyperexcitability, while the clinical diagnosis of DY TME was based on the appearance of progressive lethargy.^16–18 The incubation period was calculated as the number of days between the onset of clinical signs of the agent strain that caused disease and the inoculation of that strain. The Student''s t-test was used to compare incubation periods.¹² We found that sciatic nerve inoculation of both the HaCWD agent and 263K scrapie agent caused disease with a similar incubation period to animals infected with the HY TME agent (12 In hamsters inoculated with the DY TME agent 120 days prior to superinfection with the HaCWD or 263K agents, the animals developed clinical signs of DY TME with an incubation period that was not different from the DY TME agent control group (12 The PrP^Sc migration properties were consistent with the clinical diagnosis and all co-infected animals had PrP^Sc that migrated similar to PrP^Sc from the DY TME agent infected control animal (Fig. 1, lanes 1–10). This data indicates that the DY TME agent can interfere with more than one isolate and that interference in the CNS may be a more generalized phenomenon of prion strains.Open in a separate windowFigure 1The strain-specific properties of PrP^Sc correspond to the clinical diagnosis of disease. Western blot analysis of 250 µg brain equivalents of proteinase K digested brain homogenate from prion-infected hamsters following intracerebral (i.c.), sciatic nerve (i.sc.) or per os inoculation with either the HY TME (HY), DY TME (DY), 263K scrapie (263K), hamster-adapted CWD (CWD) agents or mock-infected (UN). The unglycoyslated PrP^Sc glycoform of HY TME, 263K scrapie and hamster-adapted CWD migrates at 21 kDa. The unglycosylated PrP^Sc glycoform of DY PrP^Sc migrates at 19 kDa. Migration of 19 and 21 kDa PrP^Sc are indicated by the arrows on the left of the figure. n.a., not applicable.

Table 1

Clinical signs and incubation periods of hamsters inoculated in the sciatic nerve with either the HY TME, HaCWD or 263K scrapie agents, or co-infected with the DY TME agent 120 days prior to superinfection of hamsters with the HY TME, HaCWD or 263K agents

Dementia screening, biomarkers and protein misfolding: Implications for public health and diagnosis

Misfolded proteins are at the core of many neurodegenerative diseases, nearly all of them associated with cognitive impairment. For example, Creutzfeldt-Jacob disease is associated with aggregation of prion protein,^1,2 Lewy body dementia and Parkinson disease with α-synuclein^3,4 and forms of frontotemporal dementia with tau, TDP43 and a host of other proteins.^5,6 Alzheimer disease (AD), the most common cause of dementia,⁷ and its prodromal syndrome mild cognitive impairment (MCI)⁸ are an increasing public health problem and a diagnostic challenge to many clinicians. AD is characterized pathologically by the accumulation of amyloid β-protein (Aβ)^9,10 as senile plaques and in the walls of blood vessels as amyloid angiopathy.^11,12 Additionally, there are accumulations of tau-protein as neurofibrillary tangles and dystrophic neurites.^11,12 Biological markers of AD and MCI can serve as in vivo diagnostic indicators of underlying pathology, particularly when clinical symptoms are mild^13–15 and are likely present years before the onset of clinical symptoms.^16–19 Research to discover and refine fluid and imaging biomarkers of protein aggregation has undergone a rapid evolution^20–22 and combined analysis of different modalities may further increase diagnostic sensitivity and specificity.^23–26 Multi-center trials are now investigating whether imaging and/or cerebrospinal fluid (CSF) biomarker candidates can be used as outcome measures for use in phase III clinical trials for AD.^27–29Key words: dementia, screening, biomarkers, amyloid, tau, Alzheimer disease, preclinical, presymptomaticCurrently, the diagnosis of AD is based on exclusion of other forms of impairment with definitive diagnosis requiring autopsy confirmation.³⁰ Thus, there is a strong need to find easily measurable in vivo AD biomarkers that could facilitate early and accurate diagnosis³¹ as well as prognostic data to assist in monitoring therapeutic efficacy.³² Although biological markers such as MRI, PET scans and CSF increase the diagnostic likelihood that AD is present,^{9,18–20,33,34} biomarkers are invasive, uncomfortable, expensive and may not be readily available to rural areas, underserved communities, underinsured individuals or developing countries, making them impractical for broad use. However, the lessons learned from biomarkers can be applied to increase the likelihood that clinicians will be able to detect disease at earlier stages in the form of dementia screening.Public health may be best defined as the organized efforts of society to improve health, often framed in terms of primary, secondary and tertiary prevention. Prevention encompasses an understanding of causation, alteration of natural history of disease and understanding of pathophysiological mechanisms.³⁵ The clearest application of this from a public health perspective is in the setting of secondary prevention (i.e., screening)—early detection as a core element, coupled with treatments or preventative actions to reduce the burden of disease.³⁵ In this instance we seek to identify individuals in whom a disease has already begun and who may be experiencing very mild clinical symptoms but have not yet sought out medical care. The objective of effective screening is to detect the disease earlier than it would have been detected with usual care. Recent healthcare reform (Accountable Care Act)³⁶ proposes a Personalized Prevention Plan including screening for cognitive disorders, reimbursable through Medicare. Thus tying knowledge about dementia screening with underlying biology of protein misfolding associated with neurodegenerative disease can have enormous implications.A review of the natural history of dementia illustrates this point (Fig. 1). The timeline of disease from presumptive start to the patient demise is plotted. Stage I marks the biologic onset of disease; however this point often cannot be identified and may begin years to decades before any evidence is apparent (represented by dashed lines). As this stage is subclinical, it is difficult to study in humans but lends itself nicely to animal models. At some point in the progression of the biology, stage II begins heralding the first pathologic evidence of disease could be obtained—in the case of AD this could include CSF measurements of amyloid and tau^22,26,27 or PET imaging with amyloid ligands.^18,37 Subsequently, the first signs and symptoms of disease develop (stage III). Till this point, the disease process has been entirely presymptomatic. Beginning with the onset of symptoms, the patient may seek medical care (stage IV) and eventually be diagnosed (stage V). From stage III onwards, the patient enters the symptomatic phase of disease. From this point, the patient is typically treated with various pharmacologic and nonpharmacologic approaches towards some outcome. Another way to envision the disease spectrum is from the biological onset to the seeking of medical attention as the preclinical phase of disease with the clinical phase beginning with the initial clinical investigations into the cause of the patients'' symptoms.Open in a separate windowFigure 1Model of the natural history of AD. Timeline from presumptive start of AD through patient diagnosis is plotted. The initiation of biological changes (stage I) marks the onset of disease and begins years to decades before any evidence is apparent (represented by dashed lines). At some point the first pathologic evidence of disease (stage II) begins and in theory can be detected with biomarkers such as CSF measurements of amyloid and tau or PET imaging with amyloid ligands. Subsequently, the first signs and symptoms of disease develop (stage III) followed by the patient seeking medical attention (stage IV) and finally a diagnosis is established (stage V). This timeline can be clustered into a presymptomatic phase (stages I–III) and a symptomatic phase (stages III–V). An alternative way to envision the disease spectrum is from the biological onset to the seeking of medical attention (stages I–IV) as the preclinical phase of disease with the clinical phase beginning with the initial clinical investigations into the cause of the patients'' symptoms (stages IV and V). Stage III is the ideal time for dementia screening.What is the value of thinking about disease in this fashion? Such models allow researchers and clinicians to model the approach to finding and applying new diagnostics and offering new interventions. From stage I to stage III, the patient is the presymptomatic, preclinical phase of disease. The only means of detection would be with a biological marker that reflected protein misfolding or some proxy marker of these events. Although longitudinal evidence of cognitive change exist from 1–3 years before clinical diagnosis, raw scores on neuropsychological testing during this time remains in the normal range.³⁸ After stage IV, the patient is in the symptomatic, clinical phase of disease. Testing here is centered on confirming the suspected diagnosis, correctly staging the disease and initiating the appropriate therapies. Basic scientific approaches focusing on the presymptomatic, preclinical phase and clinical care approaches focusing on the symptomatic, clinical phase are well established and will continue to benefit from additional research.However, if we focus only on these two phases, an opportunity will be missed to make a decidedly important impact in the patient''s well-being. From stage III to stage IV, the patient enters symptomatic, preclinical phase of disease; symptomatic because the patient or family is beginning to detect some aspect of change, but preclinical because these signs and symptoms have not yet been brought to medical attention. In the case of AD (and the other forms of dementia) this period may go for an extended length of time as patients, families and clinicians dismiss early cognitive symptoms as part of the normal aging process. Thus, the rationale for screening is that if we can identify disease earlier in its natural history than would ordinarily occur, intervention measures (those currently available and those that are being developed) would be more effective. Dementia screening therefore would be best suited to detect cognitive impairment at the beginning of disease signs (stage III), particularly if these screening measures reflect what is known about the symptomatic, clinical phase of disease and correlate with the pathologic changes occurring in the brain during the pre-symptomatic, preclinical phase of disease.In a recent paper, we evaluated the relationship between several dementia screening tests and biomarkers of AD.⁴⁰ We tested whether a reliable and validated informant-based dementia screening test (the AD8)^41,42 correlates with changes in AD biomarkers and, if positive, screening with the AD8 clinically supports an AD clinical phenotype, superior to a commonly used performance-based screening tests including the Mini Mental State Exam (MMSE)⁴³ and the Short Blessed Test (SBT).⁴⁴ A total of 257 participants were evaluated, administered a comprehensive clinical and cognitive evaluation with the Clinical Dementia Rating scale (CDR)⁴⁵ used as the gold standard. Participants consented to and completed a variety of biomarker studies including MRI, amyloid imaging using the Pittsburgh Compound B (PiB)^37,46 and CSF studies of Aβ₄₂, tau and phosphorylated tau at Serine 181 (p-tau₁₈₁).^23,24 The sample had a mean age of 75.4 ± 7.3 years with 15.1 ± 3.2 years of education. The sample was 88.7% Caucasian and 45.5% male with a mean MMSE score of 27.2 ± 3.6. The formal diagnoses of the sample was 156 CDR 0 cognitively normal, 23 CDR 0.5 MCI, 53 CDR 0.5 very mild AD and 25 CDR 1 mild AD. Participants with positive AD8 scores (graded as a score of 2 or greater) exhibited the typical AD fluid biomarker phenotype characterized by significantly lower mean levels of CSF Aβ₄₂, greater CSF tau, p-tau₁₈₁ and the tau(s)/Aβ₄₂ ratios.^26,27 They also exhibited smaller temporal lobe volumes and increased mean cortical binding potential (MCBP) for PiB imaging similar to studies of individuals with AD.^18,19 These findings support that informant-based assessments may be superior to performance-based screening measures such as the MMSE or SBT in corresponding to underlying AD pathology, particularly at the earliest stages of decline. The use of a brief test such as the AD8 may improve strategies for detecting dementia in community settings where biomarkers may not be readily available and also may enrich clinical trial recruitment by increasing the likelihood that participants have underlying biomarker abnormalities.⁴⁰To gain a better understanding of changes in biomarkers in the symptomatic, preclinical phase, a post hoc evaluation of the 156 individuals who were rated as CDR 0 no dementia at the time of their Gold Standard assessment was completed. Some of these nondemented individuals have abnormal AD biomarkers, but in the absence of performing lumbar punctures or PET scans, is it possible to detect evidence of change? AD8 scores for 132 individuals were less than 2; thus their screening test suggests no impairment (mean AD8 score = 0.30 ± 0.46). However 25 of these individuals had AD8 scores (≥2) suggesting impairment (mean AD8 score = 2.4 ± 0.91). Applying the model described in Figure 1, some of these individuals are hypothesized to be in the symptomatic, preclinical phase of disease. No difference in age, education, gender or brief performance tests (MMSE or SBT) were detected between groups (45 is increased in the individuals with higher AD8 scores supporting that informants were noticing and reporting changes in the participants cognitive function. A review of the individual AD8 questions that were first reported to change suggest that informants endorsement of subtle changes in memory (repeats questions, forgets appointments) and executive ability (trouble with judgment, appliances, finances) are valuable early signs. This is consistent with previous reports that changes in memory and judgment/problem solving CDR boxscores in nondemented individuals correlate with findings of AD pathology at autopsy.¹⁷ Although biomarkers do not reach significance in this small sample, the direction of change in favor of “Alzheimerization” of this group suggests that some of these individuals may be in the symptomatic, preclinical phase of disease. More research with larger sample sizes and longitudinal follow-up is needed to confirm this hypothesis. It should be also noted that not all individuals with an AD8 score of 2 or greater have AD. The AD8 was designed to detect cognitive impairment from all causes, and as such, these mildly affected individuals may have other causes for their cognitive change such as depression, Lewy body dementia or vascular cognitive impairment.^41,42

Table 1

Characteristics of nondemented CDR 0 individuals stratified by AD8 scores

Mouse Models of Osteoarthritis: A Summary of Models and Outcomes Assessment

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.