Stem cells in veterinary medicine--attempts at regenerating equine tendon after injury

Stem cells have evoked considerable excitement in the animal-owning public because of the promise that stem cell technology could deliver tissue regeneration for injuries for which natural repair mechanisms do not deliver functional recovery and for which current therapeutic strategies have minimal effectiveness. This review focuses on the current use of stem cells within veterinary medicine, whose practitioners have used mesenchymal stem cells (MSCs), recovered from either bone marrow or adipose tissue, in clinical cases primarily to treat strain-induced tendon injury in the horse. The background on why this treatment has been advocated, the data supporting its use and the current encouraging outcome from clinical use in horses treated with bone-marrow-derived cells are presented together with the future challenges of stem-cell therapy for the veterinary community.     

Changes in the spatial variation of soil properties following shifting cultivation in a Mexican tropical dry forest

The role of secondary vegetation in restoring soil fertility during shifting cultivation in the tropics is well known. Yet the effect of secondary succession on the spatial patterns of soil properties has received little attention. To determine whether changes in the plant community as a result of shifting cultivation affect the scale of spatial dependence for biologically important soil nutrients, we sampled three dry tropical forest stands in Campeche, Mexico. These stands represented a gradient of cultivation history: one mature forest stand, a forest fallow that had undergone one cultivation-fallow cycle, and a forest fallow that had undergone two cultivation-fallow cycles. We used an analysis of semivariance to quantify the scale and magnitude of spatial dependence for organic matter content (OM), phosphorus (P), potassium (K), and aluminum (Al) in each stand. The scale of spatial dependence varied with cultivation history, but the degree of spatial dependence did not differ among stands. In the mature forest P and K were autocorrelated over distances >7.5 m. In the forest fallows 48–88% of the variation in soil P and K was autocorrelated over distances up to 1.1–5.1 m. In contrast, the range of autocorrelation for Al (∼2.5 m) did not differ among stands. We conclude that shifting cultivation changes the range of autocorrelation for biologically important soil nutrients at a scale that may influence plant growth. The finer scaled pattern of soil nutrients in forest fallows is likely to persist with continued shifting cultivation, since fallows are cleared every 3–15 years.     

Formation of micronuclei in erythrocytes of the fathead minnow (Pimephales promelas) after acute treatment with mitomycin C or cyclophosphamide

Despite the widespread use of the fathead minnow in ecotoxicology, there have been relatively few studies on genotoxicity biomarkers in this small, warm-water fish species. Consequently, we investigated the effect of two known genotoxins, mitomycin C and cyclophosphamide, on micronucleus induction in spleen and peripheral blood erythrocytes of this species. Initially, 96-h experiments after intra-peritoneal (i.p.) injections of mitomycin C and cyclophosphamide were undertaken to determine the maximum tolerated dose (MTD). From these studies, MTDs of 10 and 400 mg/kg, respectively, were obtained: doses that were higher than those reported for other fish species. Next, an assessment of micronucleus induction at 1, 2, 4, 8 and 14 days after injection was undertaken for each compound at the MTD. Mitomycin C at 10 mg/kg significantly induced micronuclei in erythrocytes from the spleen, but not from the peripheral blood, at 8 and 14 days. In addition, the overall levels of micronuclei observed were lower than most previously published data from other fish species. In contrast to mitomycin C, treatment with 400 mg/kg cyclophosphamide failed to significantly induce micronuclei in erythrocytes from any of the tissues employed, in contrast to previous reports of significant induction in other species. The reasons for the apparent relative insensitivity of the fathead minnow to these clastogens, with respect to both MTDs and micronucleus induction, are not clear. The fathead minnow, however, has previously been described as relatively insensitive compared to other fish species with respect to selected carcinogens and cytochrome P450 inducers; the latter suggesting that the lack of a significant induction following cyclophosphamide exposure may be due to low metabolic activation in vivo. Consequently, further clarifying work is required to delineate the response shown, considering the extensive use of this species in ecotoxicology research and regulatory testing.     

The 'human revolution' in lowland tropical Southeast Asia: the antiquity and behavior of anatomically modern humans at Niah Cave (Sarawak, Borneo)

Recent research in Europe, Africa, and Southeast Asia suggests that we can no longer assume a direct and exclusive link between anatomically modern humans and behavioral modernity (the 'human revolution'), and assume that the presence of either one implies the presence of the other: discussions of the emergence of cultural complexity have to proceed with greater scrutiny of the evidence on a site-by-site basis to establish secure associations between the archaeology present there and the hominins who created it. This paper presents one such case study: Niah Cave in Sarawak on the island of Borneo, famous for the discovery in 1958 in the West Mouth of the Great Cave of a modern human skull, the 'Deep Skull,' controversially associated with radiocarbon dates of ca. 40,000 years before the present. A new chronostratigraphy has been developed through a re-investigation of the lithostratigraphy left by the earlier excavations, AMS-dating using three different comparative pre-treatments including ABOX of charcoal, and U-series using the Diffusion-Absorption model applied to fragments of bones from the Deep Skull itself. Stratigraphic reasons for earlier uncertainties about the antiquity of the skull are examined, and it is shown not to be an 'intrusive' artifact. It was probably excavated from fluvial-pond-desiccation deposits that accumulated episodically in a shallow basin immediately behind the cave entrance lip, in a climate that ranged from times of comparative aridity with complete desiccation, to episodes of greater surface wetness, changes attributed to regional climatic fluctuations. Vegetation outside the cave varied significantly over time, including wet lowland forest, montane forest, savannah, and grassland. The new dates and the lithostratigraphy relate the Deep Skull to evidence of episodes of human activity that range in date from ca. 46,000 to ca. 34,000 years ago. Initial investigations of sediment scorching, pollen, palynomorphs, phytoliths, plant macrofossils, and starch grains recovered from existing exposures, and of vertebrates from the current and the earlier excavations, suggest that human foraging during these times was marked by habitat-tailored hunting technologies, the collection and processing of toxic plants for consumption, and, perhaps, the use of fire at some forest-edges. The Niah evidence demonstrates the sophisticated nature of the subsistence behavior developed by modern humans to exploit the tropical environments that they encountered in Southeast Asia, including rainforest.     

Nutritional challenges during development induce sex-specific changes in glucose homeostasis in the adult sheep

The early-life environment has implications for risk of adult-onset diseases, such as glucose intolerance, insulin insensitivity, and obesity, effects that may occur with or without reduced birth weight. We determined the consequences of nutrient restriction in early gestation and early postnatal life and their interactions on postnatal growth, body composition, and glucose handling. Ewes received 100% (C, n = 39) or 50% nutritional requirements (U, n = 41) from 1 to 31 days gestation and 100% thereafter. Male and female offspring (singleton/twin) from C and U ewes were then fed either ad libitum (CC n = 22, UC n = 19) or to reduce body weight to 85% of target from 12 to 25 wk of age (CU n = 17, UU n = 22) and ad libitum thereafter. At 1.5 and 2.5 yr, glucose handling was determined by area under the curve (AUC) for glucose and insulin concentrations following intravenous glucose (0.5 g/kg body wt). Insulin sensitivity was determined at 2.5 yr following intravenous insulin (0.5 IU/kg). In females, postnatal undernutrition reduced (P < 0.05) glucose AUC at both ages, regardless of prenatal nutrition. Postnatal undernutrition did not affect insulin secretion in females but enhanced insulin-induced glucose disappearance in singletons. Poor early postnatal growth was associated with increased fat in females. In males, glucose tolerance was unaffected by undernutrition despite changes in insulin AUC dependent on age, treatment, and single/twin birth. Nutrition in early postnatal life has long-lasting, sex-specific effects on glucose handling in sheep, likely due, in females, to enhanced insulin sensitivity. Improved glucose utilization may aid weight recovery but have negative implications for glucose homeostasis and body composition over the longer term.     

Upregulation of PD-1 expression on circulating and intrahepatic hepatitis C virus-specific CD8+ T cells associated with reversible immune dysfunction

Infection with hepatitis C virus (HCV) is associated with persistence in the majority of individuals. We demonstrate here that the inhibitory molecule programmed death-1 (PD-1) is significantly upregulated on total and HCV-specific CD8(+) cytotoxic T lymphocytes (CTLs) in the peripheral blood and livers of patients with chronic infection compared to subjects with spontaneous HCV resolution, patients with nonviral liver disease, and normal controls. PD-1 expression on cytomegalovirus-specific CTLs also varies according to HCV status and is highest in patients with chronic infection. HCV-specific CTLs that are PD-1(high) express higher levels of the senescence marker CD57 than PD-1(low) CTLs, and CD57 expression is greater in chronic than in resolved infection. In vitro blockade of PD-1 by monoclonal antibodies specific to its ligands (PD-L1 and PD-L2) results in restoration of functional competence (proliferation and gamma interferon and interleukin-2 secretion) of HCV-specific CTLs, including those residing in the liver. This reversal of CTL exhaustion is evident even in individuals who lack HCV-specific CD4(+) T-cell help. Our data indicate that the PD-1/PD-L pathway is critical in persistent HCV infection in humans and represents a potential novel target for restoring function of exhausted HCV-specific CTLs.     

Development and Evaluation of Methods To Detect Nucleopolyhedroviruses in Larvae of the Douglas-Fir Tussock Moth, Orgyia pseudotsugata (McDunnough)

Various molecular methods are used to detect pathogenic microorganisms and viruses within their hosts, but these methods are rarely validated by direct comparison. Southern hybridization, enzyme-linked immunosorbent assay (ELISA), and a novel DNA extraction/PCR assay were used to detect Orgyia pseudotsugata multiple nucleopolyhedrovirus (OpMNPV) in Douglas-fir tussock moth larvae. PCR was more sensitive than Southern hybridization and ELISA at detecting semipurified virus. ELISA, however, was the most accurate method for detecting virus within larvae, given that Southern hybridization and PCR produced false-negative results (31% and 2.5%, respectively). ELISA may be preferable in some applications because virus infections can be quantified (r² = 0.995). These results may be applicable to both applied and academic research that seeks to accurately identify the incidence of viruses and microorganisms that regulate insect populations.     

Oxygen reactivity of both respiratory oxidases in Campylobacter jejuni: the cydAB genes encode a cyanide-resistant, low-affinity oxidase that is not of the cytochrome bd type

The microaerophilic bacterium Campylobacter jejuni is a significant food-borne pathogen and is predicted to possess two terminal respiratory oxidases with unknown properties. Inspection of the genome reveals an operon (cydAB) apparently encoding a cytochrome bd-like oxidase homologous to oxidases in Escherichia coli and Azotobacter vinelandii. However, C. jejuni cells lacked all spectral signals characteristic of the high-spin hemes b and d of these oxidases. Mutation of the cydAB operon of C. jejuni did not have a significant effect on growth, but the mutation reduced formate respiration and the viability of cells cultured in 5% oxygen. Since cyanide resistance of respiration was diminished in the mutant, we propose that C. jejuni CydAB be renamed CioAB (cyanide-insensitive oxidase), as in Pseudomonas aeruginosa. We measured the oxygen affinity of each oxidase, using a highly sensitive assay that exploits globin deoxygenation during respiration-catalyzed oxygen uptake. The CioAB-type oxidase exhibited a relatively low affinity for oxygen (K(m) = 0.8 microM) and a V(max) of >20 nmol/mg/s. Expression of cioAB was elevated fivefold in cells grown at higher rates of oxygen provision. The alternative, ccoNOQP-encoded cyanide-sensitive oxidase, expected to encode a cytochrome cb'-type enzyme, plays a major role in the microaerobic respiration of C. jejuni, since it appeared to be essential for viability and exhibited a much higher oxygen affinity, with a K(m) value of 40 nM and a V(max) of 6 to 9 nmol/mg/s. Low-temperature photodissociation spectrophotometry revealed that neither oxidase has ligand-binding activity typical of the heme-copper oxidase family. These data are consistent with cytochrome oxidation during photolysis at low temperatures.     

Creative or created: using anecdotes to investigate animal cognition

In non-human animals, creative behaviour occurs spontaneously only at low frequencies, so is typically missed by standardised observational methods. Experimental approaches have tended to rely overly on paradigms from child development or adult human cognition, which may be inappropriate for species that inhabit very different perceptual worlds and possess quite different motor capacities than humans. The analysis of anecdotes offers a solution to this impasse, provided certain conditions are met. To be reliable, anecdotes must be recorded immediately after observation, and only the records of scientists experienced with the species and the individuals concerned should be used. Even then, interpretation of a single record is always ambiguous, and analysis is feasible only when collation of multiple records shows that a behaviour pattern occurs repeatedly under similar circumstances. This approach has been used successfully to study a number of creative capacities of animals: the distribution, nature and neural correlates of deception across the primate order; the occurrence of teaching in animals; and the neural correlates of several aptitudes--in birds, foraging innovation, and in primates, innovation, social learning and tool-use. Drawing on these approaches, we describe the use of this method to investigate a new problem, the cognition of the African elephant, a species whose sheer size and evolutionary distance from humans renders the conventional methods of comparative psychology of little use. The aim is both to chart the creative cognitive capacities of this species, and to devise appropriate experimental methods to confirm and extend previous findings.     

Growth of Campylobacter jejuni on nitrate and nitrite: electron transport to NapA and NrfA via NrfH and distinct roles for NrfA and the globin Cgb in protection against nitrosative stress

Pathways of electron transport to periplasmic nitrate (NapA) and nitrite (NrfA) reductases have been investigated in Campylobacter jejuni, a microaerophilic food-borne pathogen. The nap operon is unusual in lacking napC (encoding a tetra-haem c-type cytochrome) and napF, but contains a novel gene of unknown function, napL. The iron-sulphur protein NapG has a major role in electron transfer to the NapAB complex, but we show that slow nitrate-dependent growth of a napG mutant can be sustained by electron transfer from NrfH, the electron donor to the nitrite reductase NrfA. A napL mutant possessed approximately 50% lower NapA activity than the wild type but showed normal growth with nitrate as the electron acceptor. NrfA was constitutive and was shown to play a role in protection against nitrosative stress in addition to the previously identified NO-inducible single domain globin, Cgb. However, nitrite also induced cgb expression in an NssR-dependent manner, suggesting that growth of C. jejuni with nitrite causes nitrosative stress. This was confirmed by lack of growth of cgb and nssR mutants, and slow growth of the nrfA mutant, in media containing nitrite. Thus, NrfA and Cgb together provide C. jejuni with constitutive and inducible components of a robust defence against nitrosative stress.