Huntington's disease: Dancing in a dish

In a recent landmark paper, the Huntington''s disease (HD) iPSC Consortium reports on the establishment and characterization of a panel of iPSC lines from HD patients, and more importantly, the successful modeling of HD in vitro. In the same issue of Cell Stem Cell, An et al. reports on the successful targeted gene correction of HD in human iPSCs. Both advances are exciting, provide new resources for current and future HD research, and uncover new challenges to better understand and, most importantly, treat this devastating disease in the near future.Modeling human diseases using induced pluripotent stem cells (iPSCs) has created novel opportunities for both mechanistic studies as well as for the discovery of new disease therapies. Combined with advanced gene correction technology, human iPSCs hold great promise to provide patient-specific and mutation-free cells for potential cell replacement therapy. Huntington''s disease (HD) is an autosomal dominant neurodegenerative disorder, which causes motor dysfunction, psychiatric disturbances and cognitive impairment¹. HD is caused by an expanded cystosine adenine guanine (CAG) tri-nucleotide repeat encoding polyglutamine in the first exon of the Huntingtin (HTT) gene. To date, there is no effective therapy for preventing the onset or slowdown of this disorder. Preliminary clinical trials using fetal neural grafts had shown long-lasting functional benefits in patients². Though only effective in limited cases, these results suggest that cell-based therapy could be a potential treatment if a reliable and consistent cell source is available. For this purpose, an alternative cell source to overcome the logistical and biological hurdles of this disease had been actively explored in the past decade. With recent advancement in human iPSCs technology, HD patient-specific iPSCs coupled with an efficient directed cell differentiation protocol offers hope for an unlimited supply of autologous cells. Since HD is a monogenic disease, with a very well-established correlation between the number of CAG repeats and the age of disease onset, it provides an ideal target for iPSC-based gene correction that will allow for the production of disease-free cells for potential autologous cell therapy, and at the same time provide a much needed, valuable platform to further study the pathogenesis of the disease^3,4.This is in fact what has been recently accomplished in two reports published in Cell Stem Cell^5,6. The HD iPSC Consortium reports on the generation of HD patient-specific iPSC lines that showed CAG-repeat-expansion-associated phenotypes⁵. The study from An et al.⁶ reports on the successful targeted correction of expanded CAG repeat in HD patient iPSCs and the reversion of disease phenotypes.In the study reported from the HD iPSC Consortium, the authors generated 14 iPSC lines from HD patients and controls (listed in Open in a separate window

A new HCV mouse model on the block

The investigation of virus-induced liver disease and hepatocellular carcinoma needs small animal models modeling hepatitis C virus (HCV) infection and liver disease biology. A recent study published in Cell Research reports a novel mouse model which is permissive for chronic HCV infection and shows chronic liver injury including inflammation, steatosis and fibrosis.Chronic hepatitis C virus (HCV) infection is a major cause of liver disease worldwide. The development of direct-acting antivirals has revolutionized treatment by offering cure¹. However, several hurdles remain. High costs limit treatment access in the majority of patients. Infection is often diagnosed at a late stage when advanced liver disease and cancer are established. Cure in advanced liver disease does not eliminate the risk of hepatocellular carcinoma (HCC). Re-infection remains possible and a vaccine is not available².To better understand the pathogenesis of virus-induced liver disease and HCC, a small animal model permissive for HCV infection and modeling liver disease biology is needed³. HCV infection is limited to humans and chimpanzees, predominantly due to distinct host-dependency factors and innate antiviral immune responses precluding cross-infection of other species⁴. Research efforts have focused on humanizing mice permissive to HCV infection. This has led to the development of conceptually three different types of mouse models.The human liver chimeric mouse is based on immune- and hepato-deficient mice repopulated with human hepatocytes. While the uPA-SCID⁵ and FRG⁶ models are extremely useful to study the viral life cycle and antivirals, the lack of an adaptive immune system and liver disease precludes the use for the study of liver disease biology and vaccine evaluation (7 based on modified Rag-2^−/− mice, activation of the overexpressed FK506-binding protein and caspase-8 fusion protein in the liver induces death of mouse hepatocytes and facilitates engraftment of human hepatocyte progenitor and CD34⁺ haematopoetic stem cells. While infected mice exhibit liver inflammation and fibrosis, this model appears to be limited with detection of virus only in the liver (8. Sustained and robust HCV infection for 90 days was achieved by crossing the 4hEF mice with mice knocked out for STAT1⁹. Furthermore, HCV infection in these mice elicited antiviral cellular and humoral immune responses. Although the animals were not reported to develop liver disease, this robust model represents a major breakthrough since it allows for studying HCV-induced immune responses and the preclinical evaluation of vaccine candidates in a small animal model (

Comparative Analysis of Myxococcus Predation on Soil Bacteria

Predator-prey relationships among prokaryotes have received little attention but are likely to be important determinants of the composition, structure, and dynamics of microbial communities. Many species of the soil-dwelling myxobacteria are predators of other microbes, but their predation range is poorly characterized. To better understand the predatory capabilities of myxobacteria in nature, we analyzed the predation performance of numerous Myxococcus isolates across 12 diverse species of bacteria. All predator isolates could utilize most potential prey species to effectively fuel colony expansion, although one species hindered predator swarming relative to a control treatment with no growth substrate. Predator strains varied significantly in their relative performance across prey types, but most variation in predatory performance was determined by prey type, with Gram-negative prey species supporting more Myxococcus growth than Gram-positive species. There was evidence for specialized predator performance in some predator-prey combinations. Such specialization may reduce resource competition among sympatric strains in natural habitats. The broad prey range of the Myxococcus genus coupled with its ubiquity in the soil suggests that myxobacteria are likely to have very important ecological and evolutionary effects on many species of soil prokaryotes.Predation plays a major role in shaping both the ecology and evolution of biological communities. The population and evolutionary dynamics of predators and their prey are often tightly coupled and can greatly influence the dynamics of other organisms as well (1). Predation has been invoked as a major cause of diversity in ecosystems (11, 12). For example, predators may mediate coexistence between superior and inferior competitors (2, 13), and differential trajectories of predator-prey coevolution can lead to divergence between separate populations (70).Predation has been investigated extensively in higher organisms but relatively little among prokaryotes. Predation between prokaryotes is one of the most ancient forms of predation (27), and it has been proposed that this process may have been the origin of eukaryotic cells (16). Prokaryotes are key players in primary biomass production (44) and global nutrient cycling (22), and predation of some prokaryotes by others is likely to significantly affect these processes. Most studies of predatory prokaryotes have focused on Bdellovibrionaceae species (e.g., see references 51, 55, and 67). These small deltaproteobacteria prey on other Gram-negative cells, using flagella to swim rapidly until they collide with a prey cell. After collision, the predator cells then enter the periplasmic space of the prey cell, consume the host cell from within, elongate, and divide into new cells that are released upon host cell lysis (41). Although often described as predatory, the Bdellovibrionaceae may also be considered to be parasitic, as they typically depend (apart from host-independent strains that have been observed [60]) on the infection and death of their host for their reproduction (47).In this study, we examined predation among the myxobacteria, which are also deltaproteobacteria but constitute a monophyletic clade divergent from the Bdellovibrionaceae (17). Myxobacteria are found in most terrestrial soils and in many aquatic environments as well (17, 53, 74). Many myxobacteria, including the model species Myxococcus xanthus, exhibit several complex social traits, including fruiting body formation and spore formation (14, 18, 34, 62, 71), cooperative swarming with two motility systems (64, 87), and group (or “wolf pack”) predation on both bacteria and fungi (4, 5, 8, 9, 15, 50). Using representatives of the genus Myxococcus, we tested for both intra- and interspecific variation in myxobacterial predatory performance across a broad range of prey types. Moreover, we examined whether prey vary substantially in the degree to which they support predatory growth by the myxobacteria and whether patterns of variation in predator performance are constant or variable across prey environments. The latter outcome may reflect adaptive specialization and help to maintain diversity in natural populations (57, 59).Although closely related to the Bdellovibrionaceae (both are deltaproteobacteria), myxobacteria employ a highly divergent mode of predation. Myxobacteria use gliding motility (64) to search the soil matrix for prey and produce a wide range of antibiotics and lytic compounds that kill and decompose prey cells and break down complex polymers, thereby releasing substrates for growth (66). Myxobacterial predation is cooperative both in its “searching” component (6, 31, 82; for details on cooperative swarming, see reference 64) and in its “handling” component (10, 29, 31, 32), in which secreted enzymes turn prey cells into consumable growth substrates (56, 83). There is evidence that M. xanthus employs chemotaxis-like genes in its attack on prey cells (5) and that predation is stimulated by close contact with prey cells (48).Recent studies have revealed great genetic and phenotypic diversity within natural populations of M. xanthus, on both global (79) and local (down to centimeter) scales (78). Phenotypic diversity includes variation in social compatibility (24, 81), the density and nutrient thresholds triggering development (33, 38), developmental timing (38), motility rates and patterns (80), and secondary metabolite production (40). Although natural populations are spatially structured and both genetic diversity and population differentiation decrease with spatial scale (79), substantial genetic diversity is present even among centimeter-scale isolates (78). No study has yet systematically investigated quantitative natural variation in myxobacterial predation phenotypes across a large number of predator genotypes.Given the previous discovery of large variation in all examined phenotypes, even among genetically extremely similar strains, we anticipated extensive predatory variation as well. Using a phylogenetically broad range of prey, we compared and contrasted the predatory performance of 16 natural M. xanthus isolates, sampled from global to local scales, as well as the commonly studied laboratory reference strain DK1622 and representatives of three additional Myxococcus species: M. flavescens (86), M. macrosporus (42), and M. virescens (63) (Table ​(Table1).1). In particular, we measured myxobacterial swarm expansion rates on prey lawns spread on buffered agar (31, 50) and on control plates with no nutrients or with prehydrolyzed growth substrate.

TABLE 1.

List of myxobacteria used, with geographical origin

Intra- and inter-generic transfer of pathogenicity island-encoded virulence genes by cos phages

Bacteriophage-mediated horizontal gene transfer is one of the primary driving forces of bacterial evolution. The pac-type phages are generally thought to facilitate most of the phage-mediated gene transfer between closely related bacteria, including that of mobile genetic elements-encoded virulence genes. In this study, we report that staphylococcal cos-type phages transferred the Staphylococcus aureus pathogenicity island SaPIbov5 to non-aureus staphylococcal species and also to different genera. Our results describe the first intra- and intergeneric transfer of a pathogenicity island by a cos phage, and highlight a gene transfer mechanism that may have important implications for pathogen evolution.Classically, transducing phages use the pac site-headful system for DNA packaging. Packaging is initiated on concatemeric post-replicative DNA by terminase cleavage at the sequence-specific pac site, a genome slightly longer than unit length is packaged, and packaging is completed by non-sequence-specific cleavage (reviewed in Rao and Feiss, 2008). Generalized transduction results from the initiation of packaging at pac site homologs in host chromosomal or plasmid DNA, and typically represents ∼1% of the total number of phage particles. In the alternative cos site mechanism packaging is also initiated on concatemeric post-replicative DNA by terminase cleavage at a sequence-specific (cos) site. Here, however, packaging is completed by terminase cleavage at the next cos site, generating a precise monomer with the cohesive termini used for subsequent circularization (Rao and Feiss, 2008). Although cos site homologs may exist in host DNA, it is exceedingly rare that two such sites would be appropriately spaced. Consequently, cos phages, of which lambda is the prototype, do not engage in generalized transduction. For this reason, cos-site phages have been preferred for possible phage therapy, since they would not introduce adventitious host DNA into target organisms.The Staphylococcus aureus pathogenicity islands (SaPIs) are the best-characterized members of the phage-inducible chromosomal island family of mobile genetic elements (MGEs; Novick et al., 2010). SaPIs are ∼15 kb mobile elements that encode virulence factors and are parasitic on specific temperate (helper) phages. Helper phage proteins are required to lift their repression (Tormo-Más et al., 2010, 2013), thereby initiating their excision, circularization and replication. Phage-induced lysis releases vast numbers of infectious SaPI particles, resulting in high frequencies of transfer. Most SaPI helper phages identified to date are pac phages, and many well-studied SaPIs are packaged by the headful mechanism (Ruzin et al., 2001; Ubeda et al., 2007). Recently, we have reported that some SaPIs, of which the prototype is SaPIbov5 (Viana et al., 2010), carry phage cos sequences in their genomes, and can be efficiently packaged and transferred by cos phages to S. aureus strains at high frequencies (Quiles-Puchalt et al., 2014). Here we show that this transfer extends to non-aureus staphylococci and to Listeria monocytogenes.Since the pac phages transfer SaPIs to non-aureus staphylococci and to the Gram-positive pathogen Listeria monocytogenes (Maiques et al., 2007; Chen and Novick, 2009), we reasoned that cos phages might also be capable of intra- and intergeneric transfer. We tested this with SaPIbov5, into which we had previously inserted a tetracycline resistance (tetM) marker to enable selection, and with lysogens of two helper cos phages, φ12 and φSLT, carrying SaPIbov5 (strains JP11010 and JP11194, respectively; Supplementary Table 1). The prophages in these strains were induced with mitomycin C, and the resulting lysates were adjusted to 1 μg ml⁻¹ DNase I and RNase A, filter sterilized (0.2 μm pore), and tested for SaPI transfer with tetracycline selection, as previously described (Ubeda et al., 2008). To test for trans-specific or trans-generic transduction, coagulase-negative staphylococci species and L. monocytogenes strains were used as recipients for SaPIbov5 transfer, respectively, as previously described (Maiques et al., 2007; Chen and Novick, 2009). As shown in Figure 1 and Supplementary Table 2). In contrast, deletion of the SaPIbov5 cos site (strains JP11229 and JP11230) did not affect SaPI replication (Supplementary Figure 1), but completely eliminated SaPIbov5 transfer (Supplementary Table 2). The TerS protein is essential for φ12 and SaPIbov5 DNA packaging, but not for phage-mediated lysis (Quiles-Puchalt et al., 2014). As expected, this mutation abolished SaPIbov5 transfer (Open in a separate windowFigure 1(a) Map of SaPIbov5. Arrows represent the localization and orientation of ORFs greater than 50 amino acids in length. Rectangles represent the position of the ori (in purple) or cos (in red) sites. Positions of different primers described in the text are shown. (b) Amplimers generated for detection of SaPIbov5 in the different recipient strains. Supplementary Table 2 lists the sequence of the different primers used. The element was detected in S. epidermidis JP829 (Se-1), S. epidermidis JP830 (Se-2), L. monocytogenes SK1351 (Lm-1), L. monocytogenes EGDe (Lm-2), S. xylosus C2a (Sx) and S. aureus JP4226 (Sa).

Table 1

Intra- and intergeneric SaPIbov5 transfer^a

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.

Evidence for a New Avian Paramyxovirus Serotype 10 Detected in Rockhopper Penguins from the Falkland Islands

The biological, serological, and genomic characterization of a paramyxovirus recently isolated from rockhopper penguins (Eudyptes chrysocome) suggested that this virus represented a new avian paramyxovirus (APMV) group, APMV10. This penguin virus resembled other APMVs by electron microscopy; however, its viral hemagglutination (HA) activity was not inhibited by antisera against any of the nine defined APMV serotypes. In addition, antiserum generated against this penguin virus did not inhibit the HA of representative viruses of the other APMV serotypes. Sequence data produced using random priming methods revealed a genomic structure typical of APMV. Phylogenetic evaluation of coding regions revealed that amino acid sequences of all six proteins were most closely related to APMV2 and APMV8. The calculation of evolutionary distances among proteins and distances at the nucleotide level confirmed that APMV2, APMV8, and the penguin virus all were sufficiently divergent from each other to be considered different serotypes. We propose that this isolate, named APMV10/penguin/Falkland Islands/324/2007, be the prototype virus for APMV10. Because of the known problems associated with serology, such as antiserum cross-reactivity and one-way immunogenicity, in addition to the reliance on the immune response to a single protein, the hemagglutinin-neuraminidase, as the sole base for viral classification, we suggest the need for new classification guidelines that incorporate genome sequence comparisons.Viruses from the Paramyxoviridae family have caused disease in humans and animals for centuries. Over the last 40 years, many paramyxoviruses isolated from animals and people have been newly described (16, 17, 22, 29, 31, 32, 36, 42, 44, 46, 49, 58, 59, 62-64). Viruses from this family are pleomorphic, enveloped, single-stranded, nonsegmented, negative-sense RNA viruses that demonstrate serological cross-reactivity with other paramyxoviruses related to them (30, 46). The subfamily Paramyxovirinae is divided into five genera: Respirovirus, Morbillivirus, Rubulavirus, Henipavirus, and Avulavirus (30). The Avulavirus genus contains nine distinct avian paramyxovirus (APMV) serotypes (Table ​(Table1),1), and information on the discovery of each has been reported elsewhere (4, 6, 7, 9, 12, 34, 41, 50, 51, 60, 68).

TABLE 1.

Characteristics of prototype viruses APMV1 to APMV9 and the penguin virus

Imprinted gene expression in hybrids: perturbed mechanisms and evolutionary implications

Diverse mechanisms contribute to the evolution of reproductive barriers, a process that is critical in speciation. Amongst these are alterations in gene products and in gene dosage that affect development and reproductive success in hybrid offspring. Because of its strict parent-of-origin dependence, genomic imprinting is thought to contribute to the aberrant phenotypes observed in interspecies hybrids in mammals and flowering plants, when the abnormalities depend on the directionality of the cross. In different groups of mammals, hybrid incompatibility has indeed been linked to loss of imprinting. Aberrant expression levels have been reported as well, including imprinted genes involved in development and growth. Recent studies in humans emphasize that genetic diversity within a species can readily perturb imprinted gene expression and phenotype as well. Despite novel insights into the underlying mechanisms, the full extent of imprinted gene perturbation still remains to be determined in the different hybrid systems. Here we review imprinted gene expression in intra- and interspecies hybrids and examine the evolutionary scenarios under which imprinting could contribute to hybrid incompatibilities. We discuss effects on development and reproduction and possible evolutionary implications.In many plants and animals, interspecific hybridization events yield offspring that are phenotypically different from either of the parent species. Such hybrids typically display developmental abnormalities and, in animals, often have reduced fertility or complete sterility, particularly in males. Hybrid incompatibilities arise because, although the parental species may be genetically similar, the genomes are still too divergent to sustain normal development, physiology and reproduction when mixed in the hybrid offspring (Wu and Ting, 2004). Extensive research has been performed on genetic incompatibilities in plant and animal hybrids (Ishikawa and Kinoshita, 2009; Johnson, 2010). Key loci have been mapped and characterized in experimental model species, providing important insights into the aberrant phenotypes such as male hybrid sterility (Maheshwari and Barbash, 2011).Phenotypic abnormalities in interspecies hybrids often differ greatly between the reciprocal crosses. The classic example of such an asymmetry is seen in reciprocal crosses between donkeys and horses, where both directions of the cross produce sterile offspring, but the gross phenotype of the progeny (that is, ‘mule'' versus ‘hinny'') depends on the direction of the cross. Horses and donkeys have a different chromosome number, but this cannot explain the differential hybrid phenotypes that depend on the direction of the cross (as the reciprocal crosses have the same autosomal karyotype). More than 50 years ago serum concentrations of a placental hormone were reported to be markedly higher in mule than in hinny conceptuses, suggestive of parental genome-specific gene expression (Allen, 1969).The North-American genus Peromyscus (‘deer mice'') has been studied extensively to explore hybrid incompatibilities in mammals (see Vrana et al., 1998). Also in interspecies hybrids in Mus (house mouse), between the sympatric species M. musculus and M. spretus, morphological differences are apparent between reciprocal hybrids (Zechner et al., 2004). These hybrid effects were observed in crosses between a mixed M. musculus domesticus strain and lab stocks of M. spretus. To be definitive about where the incompatibilities lie between M. musculus and M. spretus (or M. m. castaneus, see below), reciprocal crosses between several different wild-derived stocks (or wild caught animals) of M. musculus and M. spretus populations would be needed.

Table 1

Terminology and abbreviations