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) (

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

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

Influence of polyploidy on insect herbivores of native and invasive genotypes of Solidago gigantea (Asteraceae)

Herbivores are sensitive to the genetic structure of plant populations, as genetics underlies plant phenotype and host quality. Polyploidy is a widespread feature of angiosperm genomes, yet few studies have examined how polyploidy influences herbivores. Introduction to new ranges, with consequent changes in selective regimes, can lead to evolution of changes in plant defensive characteristics and also affect herbivores. Here, we examine how insect herbivores respond to polyploidy in Solidago gigantea, using plants derived from both the native range (USA) and introduced range (Europe). S. gigantea has three cytotypes in the US, with two of these present in Europe. We performed bioassays with generalist (Spodoptera exigua) and specialist (Trirhabda virgata) leaf-feeding insects. Insects were reared on detached leaves (Spodoptera) or potted host plants (Trirhabda) and mortality and mass were measured. Trirhabda larvae showed little variation in survival or pupal mass attributable to either cytotype or plant origin. Spodoptera larvae were more sensitive to both cytotype and plant origin: they grew best on European tetraploids and poorly on US diploids (high mortality) and US tetraploids (low larval mass). These results show that both cytotype and plant origin influence insect herbivores, but that generalist and specialist insects may respond differently.Key words: polyploidy, cytotype, Solidago gigantea, insect herbivore, herbivory, invasive plant, introduced plantPolyploidy, or the possession of more than two sets of homologous chromosomes, is a fundamental force in angiosperm evolution.^1,2 Many plant species or species complexes consist of multiple cytotypes that may occur sympatrically;³ this is an important source of genetic structure in plant populations that is often overlooked.⁴ Possession of multiple genomes may confer advantages to polyploid plants such as increased heterozygosity, a decreased probability of inbreeding depression, or a greater gene pool available for selection; these traits contribute to the widespread success of polyploids and may make them prone to invasiveness.^5,6 In a recent article,⁷ we examined the functional consequences of polyploidy for different cytotypes of Solidago gigantea Ait. (Asteraceae), collected from both its native range (North America) and its introduced range (Europe). In this addendum, we show how cytotype and continent of origin influence interactions of S. gigantea with insect herbivores. Interactions with herbivores are expected to vary with cytotype because of phenotypic changes associated with polyploidy, but this area has received little study (reviewed in refs. ^8–11). Plant origin, from either the native range or an introduced range, should also influence herbivores. Plants may escape from their specialist natural enemies in the introduced range, thereby experiencing reduced herbivore pressure from an insect community dominated by generalists.^12,13 Given sufficient time, plants from the introduced range may evolve to decrease investment in anti-herbivore defenses, particularly those effective against specialists.¹⁴ While a growing body of research has addressed whether plant defenses against herbivory are lower in the introduced range,^12,15,16 few of these studies have also examined the influence of cytotype.¹⁷Three cytotypes of S. gigantea can be found in its native range in North America (diploid, tetraploid and hexaploid, 2n = 18, 36 and 54 respectively). These are morphologically indistinguishable and not generally treated as separate species.¹⁸ In Europe, where S. gigantea was introduced in the mid 18^th century,¹⁹ tetraploids are the dominant cytotype but diploids also occur. S. gigantea supports a diverse array of insect herbivores in its native range, but has few natural enemies in its introduced range.²⁰ We report here on experiments using both a generalist and a specialist leaf-chewing insect. The generalist, Spodoptera exigua (Lepidoptera: Noctuidae) is widely distributed and highly polyphagous, while the specialist Trirhabda virgata (Coleoptera: Chrysomelidae) feeds only on closely-related species within the genus Solidago. T. virgata is an outbreak insect that can be a major defoliator of S. gigantea and related species in North America.²¹ We grew plants originating from 10 populations in the US and 20 populations in Europe in common gardens at the University of Wisconsin-Milwaukee Field Station in Saukville, Wisconsin. There were five plant origin-cytotype combinations: three cytotypes from the US and two from Europe. Insects were reared on detached leaves from a single plant (Spodoptera) or on potted host plants (Trirhabda), for a set period of 21 d (Spodoptera) or until pupation (Trirhabda). We recorded insect survival and mass at the end of 21 d (Spodoptera) or at pupation (Trirhabda) (reviewed in ref. ²²).Overall survival was much better for the specialist Trirhabda than for the generalist Spodoptera (91% vs. 72%). Spodoptera larvae are not generally found on S. gigantea in the field, and while they are able to complete development, we found that this plant was not an ideal host. Spodoptera larvae were more sensitive to differences among cytotype and plant origin than were Trirhabda larvae. Percent survival was particularly poor for Spodoptera larvae reared on diploids from the US, where slightly more than half of the caterpillars survived for 21 days (Fig. 1). Trirhabda pupal mass was remarkably consistent across the five ploidy-plant origin combinations. In contrast, Spodoptera larvae responded to both cytotype and continent of origin. Surviving Spodoptera larvae did particularly well on tetraploid plants from the introduced range (Europe), and particularly poorly on tetraploids from the US (Fig. 1). We have previously reported that Spodoptera grow better on plants from Europe;²² our current results reveal that this difference is due exclusively to better growth on tetraploid plants. However, our results also show that both diploids and tetraploids from the US were poor hosts for Spodoptera: diploids because they caused high mortality and tetraploids because they resulted in poor growth. These results indicate that plants from the introduced range have reduced defenses against herbivores, even when accounting for polyploidy.Open in a separate windowFigure 1Mass ± se of S. exigua (A) and T. virgata (B) larvae reared on host plants of different cytotypes of Solidago gigantea originating from the US (native range) or europe (introduced range). Means in A followed by different letters are significantly different at p < 0.05 (ANOVA followed by multiple Student''s t-tests with Bonferroni correction). There were no significant differences in (B). Sample sizes for (A and B) shown in SpodopteraTrirhabdaNo. SurvivingInitial No.% SurvivalNo. SurvivingInitial No.% SurvivalUS-Diploid213954373995US-Tetraploid709375829289US-Hexaploid162467232496EU-Diploid152365232496EU-Tetraploid1011297811412988Open in a separate windowInsects were reared on a single genotype of each cytotype-origin combination for 21 days (Spodoptera) or until pupation (Trirhabda). Sample sizes for each cytotype-origin combination vary because cytotypes were not known at the time plants were collected; these distributions represent frequencies of cytotypes in our collections.Effects of the host plant on Spodoptera were probably driven, at least in part, by changes in secondary chemistry. We have previously shown that foliar terpenoids, chemicals known to influence insect herbivores,^23,24 are affected by both cytotype and continent of origin.⁷ It is surprising that Trirhabda larvae were not more sensitive to these differences in secondary chemistry among the five ploidy-origin combinations, given that Trirhabda is known to respond to host-plant chemistry.²³ We have previously reported that Trirhabda growth does not differ on European and US plants²² and show here that accounting for cytotype does not change this conclusion. In a recent study on the closely-related Solidago altissima, Halverson et al.¹¹ reported that the effects of plant cytotype on 5 gall-making herbivores were complex and not easily characterized. All five herbivores responded to plant cytotype, but for four of the five insects the most preferred cytotype was not consistent across sites. It is possible in our study that Trirhabda were responding to cytotype at a finer scale than that examined here. There may be differences due to cytotype that shift among the populations that we sampled, and that are averaged out when examined at the continental scale. We lack sufficient replication of cytotypes within populations to test this possibility. Even so, our results reported here reveal that plant cytotype can be an important source of variation affecting insect herbivores, but that generalist and specialist insects may respond differently.     

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

The effects of amino acid composition on yeast prion formation and prion domain interactions

Yeast prions provide a powerful model system for examining prion formation and propagation in vivo. Yeast prion formation is driven primarily by amino acid composition, not by primary amino acid sequence. However, although yeast prion domains are consistently glutamine/asparagine-rich, they otherwise vary significantly in their compositions. Therefore, elucidating the exact compositional requirements for yeast prion formation has proven challenging. We have developed an in vivo method that allows for estimation of the prion propensity of each amino acid within the context of a yeast prion domain.¹ Using these values, we are able to predict the prion-propensity of various glutamine/asparagine-rich yeast domains. These results provide insight into the basis for yeast prion formation, and may aid in the discovery of additional novel prion domains. Additionally, we examined whether amino acid composition could drive interactions between heterologous glutamine/asparagine-rich proteins.² Although inefficient interactions between yeast prion domains have previously been observed, we found that one prion protein, Ure², is able to interact with compositionally similar domains with unprecedented efficiency. This observation, combined with the growing number of yeast prions, suggests that a broad network of interactions between heterologous glutamine/asparagine-rich proteins may affect yeast prion formation.Key words: yeast, prion, amyloid, Ure2, Sup35The highly-studied yeast prion proteins Sup35, Ure2 and Rnq1, which form the [PSI⁺], [URE3] and [PIN⁺] prions, respectively, each contain a glutamine/asparagine (Q/N) rich domain that drives prion formation. ³ Prion formation is thought to involve conversion of the soluble proteins into an insoluble amyloid form.³ Four additional yeast prion proteins containing Q/N-rich prion-forming domains have recently been discovered, and domains with similar Q/N-content are over-represented in various eukaryotic genomes.^4–8 This suggests that numerous other prion proteins may remain to be discovered, yet predicting which of these Q/N-rich domains is likely to form prions has proven difficult.Randomizing the order of the amino acids in either the Sup35 or Ure2 prion domains does not block prion formation, demonstrating that prion formation is driven primarily by amino acid composition, not primary sequence.^9,10 We therefore explored two related questions. First, can amino acid composition be used to accurately predict prion propensity?¹ Second, to what extent can compositionally similar Q/N-rich proteins interact during prion formation?²Surprisingly, although composition drives yeast prion formation, compositional similarity to known prion domains is a poor predictor of prion propensity. In addition to high Q/N content, yeast prion domains show an under-representation of both charged and hydrophobic residues (1^,11 Alberti et al. recently identified the 100 yeast domains with greatest compositional similarity to the known prion domains, and tested each domain in four different assays for prion-like activity.⁴ This remarkable effort revealed numerous new potential prion domains; however, there was very little correlation between a domain’s compositional similarity to known prion domains and its prion-like activity.^1,4 The most likely explanation for this unexpected finding is that because the yeast prion domains are likely not optimized for maximum prion propensity, some compositional deviations will increase prion propensity and some will decrease it. However, algorithms that predict prion propensity based on compositional similarity to known prion domains are predicated on the assumption that all compositional changes will reduce prion propensity. Instead, accurate prediction of prion propensity requires understanding how changes in amino acid composition affect prion propensity.

Table 1

Prion propensity, order propensity and prevalence for each amino acid

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

Allelic frequency and genotypes of prion protein at codon 136 and 171 in Iranian Ghezel sheep breeds

PrP genotypes at codons 136 and 171 in 120 Iranian Ghezel sheep breeds were studied using allele-specific PCR amplification and compared with the well-known sheep breeds in North America, the United States and Europe. The frequency of V allele and VV genotype at codon 136 of Ghezel sheep breed was significantly lower than AA and AV. At codon 171, the frequency of allele H was significantly lower than Q and R. Despite the similarities of PrP genotypes at codons 136 and 171 between Iranian Ghezel sheep breeds and some of the studied breeds, significant differences were found with others. Planning of effective breeding control and successful eradication of susceptible genotypes in Iranian Ghezel sheep breeds will not be possible unless the susceptibility of various genotypes in Ghezel sheep breeds to natural or experimental scrapie has been elucidated.Key words: scrapie, Ghezel sheep breed, PrP genotyping, allele specific amplification, codon 136, codon 171Scrapie was first described in England in 1732,¹ and it is an infectious neurodegenerative fatal disease of sheep and goats belonging to the group of transmissible subacute spongiform encephalopathies (TSEs), along with bovine spongiform encephalopathy (BSE), chronic wasting disease and Creutzfeldt-Jakob disease.^2,3 The term prion, proteinaceous infectious particles, coined by Stanley B. Prusiner, was introduced, and he presents the idea that the causal agent is a protein.⁴ Prion proteins are discovered in two forms, the wild-type form (PrP^c) and the mutant form (PrP^Sc).⁵ Although scrapie is an infectious disease, the susceptibility of sheep is influenced by genotypes of the prion protein (PrP) gene.^2,6 Researchers have found that the PrP allelic variant alanine/arginine/arginine (ARR) at codons 136, 154 and 171 is associated with resistance to scrapie in several breeds.^7–14 Most of the sheep populations in the Near East and North African Region (84% of the total population of 255 million) are raised in Iran, Turkey, Pakistan, Sudan, Algeria, Morocco, Afghanistan, Syria and Somalia.¹⁵ In 2003, the Iranian sheep population was estimated at 54,000,000 head. The Ghezel sheep breed, which also is known as Kizil-Karaman, Mor-Karaman, Dugli, Erzurum, Chacra, Chagra, Chakra, Gesel, Gezel, Kazil, Khezel, Khizel, Kizil, Qezel, Qizil and Turkish Brown, originated in northwestern Iran and northeastern Turkey. By considering sheep breeds as one of the main sources of meat, dairy products and related products, a global screening attempt is started in different areas. In compliance with European Union Decision 2003/100/EC, each member state has introduced a breeding program to select for resistance to TSEs in sheep populations to increase the frequency of the ARR allele. A similar breeding program is established in United States and Canada. The Near East and North African Region still needs additional programs to help the global plan of eradication of scrapie-susceptible genotypes. The current study was the first to assess the geographical and molecular variation of codons 136 and 171 polymorphism between Iranian Ghezel sheep breed and well-known sheep breeds.Polymorphism at codon 136 is associated with susceptibility to scrapie in both experimental and natural models.^10,11,13,16 17 and Austrian Carynthian sheep.¹⁸ Swiss White Alpine showed higher frequency of allele V at position 136 than Swiss Oxford Down, Swiss Black-Brown Mountain and Valais Blacknose.¹⁹ Comparison of polymorphism at codon 136 in the current study with some of other breeds (20 some flock of Hampshire sheep²¹ with current study, but the frequency of it is higher than that of some other breeds.

Table 1

Comparison of PrP allelic and genotype frequencies at codon 136 in different breeds

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

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.