Archaeology of Eukaryotic DNA Replication

Recent advances in the characterization of the archaeal DNA replication system together with comparative genomic analysis have led to the identification of several previously uncharacterized archaeal proteins involved in replication and currently reveal a nearly complete correspondence between the components of the archaeal and eukaryotic replication machineries. It can be inferred that the archaeal ancestor of eukaryotes and even the last common ancestor of all extant archaea possessed replication machineries that were comparable in complexity to the eukaryotic replication system. The eukaryotic replication system encompasses multiple paralogs of ancestral components such that heteromeric complexes in eukaryotes replace archaeal homomeric complexes, apparently along with subfunctionalization of the eukaryotic complex subunits. In the archaea, parallel, lineage-specific duplications of many genes encoding replication machinery components are detectable as well; most of these archaeal paralogs remain to be functionally characterized. The archaeal replication system shows remarkable plasticity whereby even some essential components such as DNA polymerase and single-stranded DNA-binding protein are displaced by unrelated proteins with analogous activities in some lineages.Double-stranded DNA is the molecule that carries genetic information in all cellular life-forms; thus, replication of this genetic material is a fundamental physiological process that requires high accuracy and efficiency (Kornberg and Baker 2005). The general mechanism and principles of DNA replication are common in all three domains of life—archaea, bacteria, and eukaryotes—and include recognition of defined origins, melting DNA with the aid of dedicated helicases, RNA priming by the dedicated primase, recruitment of DNA polymerases and processivity factors, replication fork formation, and simultaneous replication of leading and lagging strands, the latter via Okazaki fragments (Kornberg and Baker 2005; Barry and Bell 2006; Hamdan and Richardson 2009; Hamdan and van Oijen 2010). Thus, it was a major surprise when it became clear that the protein machineries responsible for this complex process are drastically different, especially in bacteria compared with archaea and eukarya. The core components of the bacterial replication systems, such as DNA polymerase, primase, and replication helicase, are unrelated or only distantly related to their counterparts in the archaeal/eukaryotic replication apparatus (Edgell 1997; Leipe et al. 1999).The existence of two distinct molecular machines for genome replication has raised obvious questions on the nature of the replication system in the last universal common ancestor (LUCA) of all extant cellular life-forms, and three groups of hypotheses have been proposed (Leipe et al. 1999; Forterre 2002; Koonin 2005, 2006, 2009; Glansdorff 2008; McGeoch and Bell 2008): (1) The replication systems in Bacteria and in the archaeo–eukaryotic lineage originated independently from an RNA-genome LUCA or from a noncellular ancestral state that encompassed a mix of genetic elements with diverse replication strategies and molecular machineries. (2) The LUCA was a typical cellular life-form that possessed either the archaeal or the bacterial replication apparatus in which several key components have been replaced in the other major cellular lineage. (3) The LUCA was a complex cellular life-form that possessed both replication systems, so that the differentiation of the bacterial and the archaeo–eukaryotic replication machineries occurred as a result of genome streamlining in both lines of descent that was accompanied by differential loss of components. With regard to the possible substitution of replication systems, a plausible mechanism could be replicon takeover (Forterre 2006; McGeoch and Bell 2008). Under the replicon takeover hypothesis, mobile elements introduce into cells a new replication system or its components, which can displace the original replication system through one or several instances of integration of the given element into the host genome accompanied by inactivation of the host replication genes and/or origins of replication. This scenario is compatible with the experimental results showing that DNA replication DNA in Escherichia coli with an inactivated DnaAgene or origin of replication can be rescued by the replication apparatus of R1 or F1 plasmids integrated into the bacterial chromosome (Bernander et al. 1991; Koppes 1992). Furthermore, genome analysis suggests frequent replicon fusion in archaea and bacteria (McGeoch and Bell 2008); in particular, such events are implied by the observation that in archaeal genomes, genes encoding multiple paralogs of the replication helicase MCM and origins of replication are associated with mobile elements (Robinson and Bell 2007; Krupovic et al. 2010). Replicon fusion also is a plausible path from a single origin of replication that is typical of bacteria to multiple origins present in archaea and eukaryotes. However, all the evidence in support of frequent replicon fusion and the plausibility of replicon takeover notwithstanding, there is no evidence of displacement of the bacterial replication apparatus with the archaeal version introduced by mobile elements, or vice versa, displacement of the archaeal machinery with the bacterial version, despite the rapid accumulation of diverse bacterial and archaeal genome sequences. Thus, the displacement scenarios of DNA replication machinery evolution are so far not supported by comparative genomic data.Regardless of the nature of the DNA replication system (if any) in the LUCA and the underlying causes of the archaeo–bacterial dichotomy of replication machineries, the similarity between the archaeal and eukaryotic replication systems is striking (Leipe et al. 1999; Bell and Dutta 2002; Bohlke et al. 2002; Kelman and White 2005; Barry and Bell 2006). Thus, the archaeal replication system appears to be an ancestral version of the eukaryotic system and hence a good model for functional and structural studies aimed at gaining mechanistic insights into eukaryotic replication.

Table 1.

The relationship between archaeal and eukaryotic replication systems

Noncanonical Sites of Adult Neurogenesis in the Mammalian Brain

Two decades after the discovery that neural stem cells (NSCs) populate some regions of the mammalian central nervous system (CNS), deep knowledge has been accumulated on their capacity to generate new neurons in the adult brain. This constitutive adult neurogenesis occurs throughout life primarily within remnants of the embryonic germinal layers known as “neurogenic sites.” Nevertheless, some processes of neurogliogenesis also occur in the CNS parenchyma commonly considered as “nonneurogenic.” This “noncanonical” cell genesis has been the object of many claims, some of which turned out to be not true. Indeed, it is often an “incomplete” process as to its final outcome, heterogeneous by several measures, including regional location, progenitor identity, and fate of the progeny. These aspects also strictly depend on the animal species, suggesting that persistent neurogenic processes have uniquely adapted to the brain anatomy of different mammals. Whereas some examples of noncanonical neurogenesis are strictly parenchymal, others also show stem cell niche-like features and a strong link with the ventricular cavities. This work will review results obtained in a research field that expanded from classic neurogenesis studies involving a variety of areas of the CNS outside of the subventricular zone (SVZ) and subgranular zone (SGZ). It will be highlighted how knowledge concerning noncanonical neurogenic areas is still incomplete owing to its regional and species-specific heterogeneity, and to objective difficulties still hampering its full identification and characterization.The central nervous system (CNS) of adult mammals is assembled during developmental neurogenesis, and its architectural specificity is maintained through a vast cohort of membrane-bound and extracellular matrix molecules (Gumbiner 1996; Bonfanti 2006). Although CNS structure is sculpted by experience-dependent synaptic plasticity at different postnatal developmental stages (critical periods) (see Sale et al. 2009) and, to a lesser extent, during adulthood (Holtmaat and Svoboda 2009), the neural networks are rather stabilized in the “mature” nervous tissue (Spolidoro et al. 2009). The differentiated cellular elements forming adult neural circuitries remain substantially unchanged in terms of their number and types, because cell renewal/addition in the CNS is very low. This situation is intuitive because connectional, neurochemical, and functional specificities are fundamental features of the mature CNS in highly complex brains, allowing specific cell types to be connected and to act in a relatively invariant way (Frotscher 1992).Since the discovery of neural stem cells (NSCs) (Reynolds and Weiss 1992), we realized that the aforementioned rules of CNS stability have a main exception in two brain regions: the forebrain subventricular zone (SVZ) (Lois and Alvarez-Buylla 1994) and the hippocampal subgranular zone (SGZ) (Gage 2000). These “adult neurogenic sites” are remnants of the embryonic germinal layers (although indirectly for the SGZ, which forms ectopically from the embryonic germinative matrix), which retain stem/progenitor cells within a special microenvironment, a “niche,” allowing and regulating NSC activity (Kriegstein and Alvarez-Buylla 2009). In addition, the areas of destination (olfactory bulb and dentate gyrus) reached by neuroblasts generated within these neurogenic sites harbor specific, not fully identified yet, environmental signals allowing the integration of young, newborn neurons. These two “canonical” sites of adult neurogenesis have been found in all animal species studied so far, including humans (reviewed in Lindsey and Tropepe 2006; Bonfanti and Ponti 2008; Kempermann 2012; Grandel and Brand 2013). Although in several classes of vertebrates including fish, amphibians, and reptiles, adult neurogenesis is widespread in many areas of the CNS (Zupanc 2006; Chapouton et al. 2007; Grandel and Brand 2013), in mammals, the vast majority of the brain and spinal cord regions out of the germinal-layer-derived neurogenic sites are commonly referred to as “nonneurogenic parenchyma” (Sohur et al. 2006; Bonfanti and Peretto 2011; Bonfanti and Nacher 2012). However, this viewpoint has changed during the last few years. New examples of cell genesis, involving both neurogenesis and gliogenesis, have been shown to occur in the so-called nonneurogenic regions of the mammalian CNS (Horner et al. 2000; Dayer et al. 2005; Kokoeva et al. 2005; Luzzati et al. 2006; Ponti et al. 2008; reviewed in Butt et al. 2005; Nishiyama et al. 2009; Migaud et al. 2010; Bonfanti and Peretto 2011), suggesting that structural plasticity involving de novo neural cell genesis could be more widespread than previously thought. Apart from their temporal persistence (some of them represent examples of delayed developmental neurogenesis, which persist postnatally; see below), neurogliogenic processes vary as to their regional localization, origin, and final outcome. In this review, “noncanonical” neurogenic processes occurring in adult mammals will be reviewed by underlining their heterogeneity across the species and their differences in intensity and outcome with respect to canonical neurogenic sites.

Table 1.

Main sites of noncanonical neurogenesis in the mammalian brain

Cell Biology of Mitotic Recombination

Homologous recombination provides high-fidelity DNA repair throughout all domains of life. Live cell fluorescence microscopy offers the opportunity to image individual recombination events in real time providing insight into the in vivo biochemistry of the involved proteins and DNA molecules as well as the cellular organization of the process of homologous recombination. Herein we review the cell biological aspects of mitotic homologous recombination with a focus on Saccharomyces cerevisiae and mammalian cells, but will also draw on findings from other experimental systems. Key topics of this review include the stoichiometry and dynamics of recombination complexes in vivo, the choreography of assembly and disassembly of recombination proteins at sites of DNA damage, the mobilization of damaged DNA during homology search, and the functional compartmentalization of the nucleus with respect to capacity of homologous recombination.Homologous recombination (HR) is defined as the homology-directed exchange of genetic information between two DNA molecules (Fig. 1). Mitotic recombination is often initiated by single-stranded DNA (ssDNA), which can arise by several avenues (Mehta and Haber 2014). They include the processing of DNA double-strand breaks by 5′ to 3′ resection, during replication of damaged DNA, or during excision repair (Symington 2014). The ssDNA is bound by replication protein A (RPA) to control its accessibility to the Rad51 recombinase (Sung 1994, 1997a; Sugiyama et al. 1997; Morrical 2014). The barrier to Rad51-catalyzed recombination imposed by RPA can be overcome by a number of mediators, such as BRCA2 and Rad52, which serve to replace RPA with Rad51 on ssDNA, and the Rad51 paralogs Rad55-Rad57 (RAD51B-RAD51C-XRCC2-XRCC3) and the Psy3-Csm2-Shu1-Shu2 complex (SHU) (RAD51D-XRCC2-SWS1), which stabilize Rad51 filaments on ssDNA (see Sung 1997b; Sigurdsson et al. 2001; Martin et al. 2006; Bernstein et al. 2011; Liu et al. 2011; Qing et al. 2011; Amunugama et al. 2013; Zelensky et al. 2014). The Rad51 nucleoprotein filament catalyzes the invasion into a homologous duplex to produce a displacement loop (D-loop) (Fig. 1). At this stage, additional antirecombination functions are exerted by Srs2 (FBH1, PARI), which dissociates Rad51 filaments from ssDNA, and Mph1 (FANCM), which disassembles D-loops (see Daley et al. 2014). Upon Rad51-catalyzed strand invasion, the ATP-dependent DNA translocase Rad54 enables the invading 3′ end to be extended by DNA polymerases to copy genetic information from the intact duplex (Li and Heyer 2009). Ligation of the products often leads to joint molecules (JMs), such as single- or double-Holliday junctions (s/dHJs) or hemicatenanes (HCs), which must be processed to allow separation of the sister chromatids during mitosis. JMs can be dissolved by the Sgs1-Top3-Rmi1 complex (STR) (BTR, BLM-TOP3α-RMI1-RMI2) (see Bizard and Hickson 2014) or resolved by structure-selective nucleases, such as Mus81-Mms4 (MUS81-EME1), Slx1-Slx4, and Yen1 (GEN1) (see Wyatt and West 2014). Mitotic cells favor recombination events that lead to noncrossover events likely to avoid potentially detrimental consequences of loss of heterozygosity and translocations.Open in a separate windowFigure 1.Primary pathways for homology-dependent double-strand break (DSB) repair. Recombinational repair of a DSB is initiated by 5′ to 3′ resection of the DNA end(s). The resulting 3′ single-stranded end(s) invade an intact homologous duplex (in red) to prime DNA synthesis. For DSBs that are repaired by the classical double-strand break repair (DSBR) model, the displaced strand from the donor duplex pairs with the 3′ single-stranded DNA (ssDNA) tail at the other side of the break, which primes a second round of DNA synthesis. After ligation of the newly synthesized DNA to the resected 5′ strands, a double-Holliday junction (dHJ) intermediate is generated. The dHJ can be either dissolved by branch migration (indicated by arrows) into a hemicatenane (HC) leading to noncrossover (NCO) products or resolved by endonucleolytic cleavage (indicated by triangles) to produce NCO (positions 1, 2, 3, and 4) or CO (positions 1, 2, 5, and 6) products. Alternatively to the double-strand break repair (DSBR) pathway, the invading strand is often displaced after limited synthesis and the nascent complementary strand anneals with the 3′ single-stranded tail of the other end of the DSB. After fill-in synthesis and ligation, this pathway generates NCO products and is referred to as synthesis-dependent strand annealing (SDSA).

Table 1.

Evolutionary conservation of homologous recombination proteins between Saccharomyces cerevisiae and Homo sapiens

Heparan Sulfate Proteoglycans

Heparan sulfate proteoglycans are found at the cell surface and in the extracellular matrix, where they interact with a plethora of ligands. Over the last decade, new insights have emerged regarding the mechanism and biological significance of these interactions. Here, we discuss changing views on the specificity of protein–heparan sulfate binding and the activity of HSPGs as receptors and coreceptors. Although few in number, heparan sulfate proteoglycans have profound effects at the cellular, tissue, and organismal level.Heparan sulfate proteoglycans (HSPGs) are glycoproteins, with the common characteristic of containing one or more covalently attached heparan sulfate (HS) chains, a type of glycosaminoglycan (GAG) (Esko et al. 2009). Cells elaborate a relatively small set of HSPGs (∼17) that fall into three groups according to their location: membrane HSPGs, such as syndecans and glycosylphosphatidylinositol-anchored proteoglycans (glypicans), the secreted extracellular matrix HSPGs (agrin, perlecan, type XVIII collagen), and the secretory vesicle proteoglycan, serglycin (Esko et al. 1985), which allowed functional studies in the context of a cell culture model (Zhang et al. 2006). A decade later, the first HSPG mutants in a model organism (Drosophila melanogaster) were identified (Rogalski et al. 1993; Nakato et al. 1995; Häcker et al. 1997; Bellaiche et al. 1998; Lin et al. 1999), which was followed by identification of mutants in nematodes, tree frogs, zebrafish, and mice (​and3).3). HS is evolutionarily ancient and its composition has remained relatively constant from Hydra to humans (Yamada et al. 2007; Lawrence et al. 2008).

Table 1.

Heparan sulfate proteoglycans

Wnt Pathway Regulation of Embryonic Stem Cell Self-Renewal

Embryonic stem cells (ESCs) can generate all of the cell types found in the adult organism. Remarkably, they retain this ability even after many cell divisions in vitro, as long as the culture conditions prevent differentiation of the cells. Wnt signaling and β-catenin have been shown to cause strong effects on ESCs both in terms of stimulating the expansion of stem cells and stimulating differentiation toward lineage committed cell types. The varied effects of Wnt signaling in ESCs, alongside the sometimes unconventional mechanisms underlying the effects, have generated a fair amount of controversy and intrigue regarding the role of Wnt signaling in pluripotent stem cells. Insights into the mechanisms of Wnt function in stem cells can be gained by examination of the causes for seemingly opposing effects of Wnt signaling on self-renewal versus differentiation.For a single-cell embryo to eventually form an adult organism of trillions of cells, some cells in the early mammalian embryo must be able to generate all cell lineages in the animal. The potential to make all adult cell types defines the property of pluripotency, and it is maintained in proliferating cells through a process called self-renewal. As cells become specified to contribute to particular lineages, they typically lose the ability to make cell types from distinct lineages (Waddington 1957; Hochedlinger and Plath 2009). As such, pluripotency is lost during the initial steps of lineage commitment that occur during gastrulation (Beddington 1982, 1983; Lawson and Pedersen 1987; Lawson et al. 1991), which is a process that coordinates the generation of adult cell lineages with the elaboration of a basic three-dimensional body structure (Heisenberg and Solnica-Krezel 2008). In the mouse, pluripotency can be tested with various experiments; the gold standard is the injection of cells into a blastocyst-staged embryo followed by contribution to a diversity of cell types in the chimeric animal or chimeric embryo after gastrulation. Cells are typically considered to have been pluripotent only if they contributed to all three germ layers (endoderm, mesoderm, and ectoderm).Embryonic stem cells (ESCs) are generated in vitro by outgrowths from a preimplantation-staged embryo, frequently a blastocyst. Pluripotent cells from the inner cell mass (ICM) of the blastocyst proliferate to form colonies, which can be expanded into ESC cultures. When culture conditions for in vitro propagation of mouse ESCs (mESCs) were first discovered more than 30 years ago (Evans and Kaufman 1981; Martin 1981), the critical achievement was finding conditions supporting indefinite ESC self-renewal, that is, maintenance of pluripotency following cell division. Compared with the other cell systems discussed below in this article, mESCs ostensibly display the greatest capacity for self-renewal and the highest ability to maintain pluripotency. As such, mESCs are typically thought to represent a primitive, or “naive,” cellular state in the early embryo.Several culture conditions can support self-renewal of mESCs. Initially, ESCs were grown in serum containing media atop a layer of mitotically inactivated fibroblasts, called feeder cells (Evans and Kaufman 1981). Feeder cells secrete the LIF cytokine, which binds a transmembrane receptor complex consisting of LIFR and gp130 proteins (Gearing et al. 1991; Gearing and Bruce 1992; Davis et al. 1993). LIF binding activates Jak/Stat signaling and Stat3 phosphorylation, which promotes ESC self-renewal (Niwa et al. 1998; Matsuda et al. 1999). Convincing proof of LIF’s importance for self-renewal in vitro was shown when recombinant LIF protein was shown to be sufficient to replace feeder cells in ESC cultures (Smith et al. 1988; Williams et al. 1988; Nichols et al. 1990).Essentially the same feeder cells can be used for both mESCs and human ESCs (hESCs); however, discrete activities of the feeders in terms of the cytokines they release are needed to effect optimal self-renewal for each cell. The LIF cytokine important for mESC self-renewal did not stimulate hESC self-renewal (Thomson et al. 1998). Instead, ERK signaling downstream from Fgf2 must accompany a feeder layer in serum-containing media for optimal hESC self-renewal (Xu et al. 2005). Interestingly, recombinant Fgf2 by itself could not replace feeders, and Fgf2 has been suggested to work in part by stimulating feeders to produce Activin/Nodal ligands; the combination of Fgf2 and Nodal/Activin is sufficient to support hESC self-renewal in serum-free chemically defined culture conditions (Vallier et al. 2004, 2009; James et al. 2005).Clear differences exist between mESCs and hESCs. The colonies adopt different morphologies, they require distinct culture conditions for self-renewal, and they have significantly different gene expression signatures (Brons et al. 2007; Tesar et al. 2007). Mouse EpiSCs are made from the epiblast of postimplantation-staged embryos between embryonic days 5.5 (E5.5) and E6.5 of embryogenesis (Brons et al. 2007; Tesar et al. 2007; Han et al. 2010). Lineage specification of pluripotent epiblast cells begins soon after formation of a cup-like structure, and at E6.5, the cells in the epiblast begin to be specified to primary cell lineages during gastrulation. The in vivo cellular environment for ICM cells and postimplantation epiblast cells is considerably different, and it is not surprising that EpiSCs and mESCs display many different characteristics (Xu et al. 2010). However, it was somewhat surprising that EpiSCs share many characteristics with hESCs, including a common colony morphology, Fgf2 + Activin A culture conditions, and gene expression signatures (Brons et al. 2007; Tesar et al. 2007). Like mESCs and hESCs, EpiSCs pass pluripotency tests for in vitro differentiation and teratoma formation. Whereas mESC can efficiently convert (i.e., differentiate) into EpiSC-like cells when switched to Fgf2/Activin A media (Hanna et al. 2009; Greber et al. 2010), EpiSCs required genetic manipulation or reprogramming for efficient conversion to mES-like cells (Guo et al. 2009; Hanna et al. 2009; Greber et al. 2010; Guo and Smith 2010). Many investigators consider hESCs and mouse EpiSCs to be primed for differentiation as they reside in a less primitive differentiation state relative to the naive state of pluripotency in mESCs.

Table 1.

Pluripotent stem cell states: Naive and primed

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

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

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

Biological Activity of Nerve Growth Factor Precursor Is Dependent upon Relative Levels of Its Receptors

Nerve growth factor (NGF) is produced as a precursor called pro-nerve growth factor (proNGF), which is secreted by many tissues and is the predominant form of NGF in the central nervous system. In Alzheimer disease brain, cholinergic neurons degenerate and can no longer transport NGF as efficiently, leading to an increase in untransported NGF in the target tissue. The protein that accumulates in the target tissue is proNGF, not the mature form. The role of this precursor is controversial, and both neurotrophic and apoptotic activities have been reported for recombinant proNGFs. Differences in the protein structures, protein expression systems, methods used for protein purification, and methods used for bioassay may affect the activity of these proteins. Here, we show that proNGF is neurotrophic regardless of mutations or tags, and no matter how it is purified or in which system it is expressed. However, although proNGF is neurotrophic under our assay conditions for primary sympathetic neurons and for pheochromocytoma (PC12) cells, it is apoptotic for unprimed PC12 cells when they are deprived of serum. The ratio of tropomyosin-related kinase A to p75 neurotrophin receptor is low in unprimed PC12 cells compared with primed PC12 cells and sympathetic neurons, altering the balance of proNGF-induced signaling to favor apoptosis. We conclude that the relative level of proNGF receptors determines whether this precursor exhibits neurotrophic or apoptotic activity.Nerve growth factor (NGF)³ regulates neuronal survival, neurite outgrowth, and differentiation in the peripheral and central nervous systems (1). The mature form of NGF forms a non-covalent homodimer and binds with high affinity (k_d ≈ 10⁻¹¹ m) to tropomyosin-related kinase A (TrkA) and with low affinity (k_d ≈ 10⁻⁹ m) to the common neurotrophin receptor p75^NTR (p75 neurotrophin receptor) (2). NGF promotes cell survival and growth in cells expressing TrkA through activation of the phosphatidylinositol 3-kinase/AKT pathway and the Ras/mitogen-activated protein kinase (MAPK) pathway (3, 4). p75^NTR plays diverse roles, ranging from cell survival to cell death depending on the cellular context in which it is expressed. Through activation of the NF-κB pathway, p75^NTR can contribute to cell survival in sensory neurons (5), it is involved in axonal growth via regulation of Rho activity (6), and it can interact with Trks to enhance neurotrophin affinity (at low concentration of ligand) and specificity of binding to Trks (7–9). High levels of p75^NTR expression can induce apoptosis when there are low levels of Trk or when Trk is absent (10, 11). Apoptosis occurs through increased ceramide production (12), activation of c-Jun N-terminal kinase (JNK1), and p53 (10, 13). p75^NTR requires a co-receptor called sortilin to induce cell death (14).NGF is produced as a precursor called pro-nerve growth factor (proNGF) (15). ProNGF is secreted by many tissues such as prostate cells, spermatids, hair follicles, oral mucosal keratinocytes, sympathetic neurons, cortical astrocytes, heart, and spleen (16–20). ProNGF is the predominant form of NGF in the central and peripheral nervous systems, whereas little or no mature NGF can be detected (21–24). In Alzheimer disease brain, retrograde transport from the cortex and hippocampus to basal forebrain cholinergic neurons is reduced as these neurons degenerate, with concomitant proNGF accumulation in the cortex and hippocampus (21, 23). This suggested that proNGF mediates biological activity besides its prodomain function of promoting protein folding and regulation of neurotrophin secretion (25–28). To study the role of proNGF protein in vitro, point mutations were inserted at the cleavage site used by furin, a proprotein convertase known to cleave proNGF (29), to minimize the conversion of proNGF to mature NGF. The resulting recombinant, cleavage-resistant proNGFs reportedly exhibit either apoptotic activity (30, 31) or neurotrophic activity (32, 33). These recombinant proteins differ in several ways (