Introduction. RIFLE and AKIN provide a standardised classification of acute kidney injury (AKI), but their categorical rather than continuous nature restricts their use to a research tool. A more accurate real-time description of renal function in AKI is needed, and some published data suggest that equations based on serum creatinine that estimate glomerular filtration rate (eGFR) can provide this. In addition, incorporating serum cystatin C concentration into estimates of GFR may improve their accuracy, but no eGFR equations are validated in critically ill patients with AKI. Aim. This study tests whether creatinine or cystatin-C-based eGFR equations, used in patients with CKD, offer an accurate representation of 4-hour creatinine clearance (4CrCl) in critically ill patients with AKI. Methods. Fifty-one critically ill patients with AKI were recruited. Thirty-seven met inclusion criteria, and the performance of eGFR equations was compared to 4CrCl. Results. eGFR equations were better than creatinine alone at predicting 4CrCl. Adding cystatin C to estimates did not improve the bias or add accuracy. The MDRD 7 eGFR had the best combination of correlation, bias, percentage error and accuracy. None were near acceptable standards quoted in patients with chronic kidney disease (CKD). Conclusions. eGFR equations are not sufficiently accurate for use in critically ill patients with AKI. Incorporating serum cystatin C does not improve estimates. eGFR should not be used to describe renal function in patients with AKI. Standards of accuracy for validating eGFR need to be set.
Infectious disease, especially virulent infectious disease, is commonly regarded as a cause of fluctuation or decline in biological populations. However, it is not generally considered as a primary factor in causing the actual endangerment or extinction of species. We review here the known historical examples in which disease has, or has been assumed to have had, a major deleterious impact on animal species, including extinction, and highlight some recent cases in which disease is the chief suspect in causing the outright endangerment of particular species. We conclude that the role of disease in historical extinctions at the population or species level may have been underestimated. Recent methodological breakthroughs may lead to a better understanding of the past and present roles of infectious disease in influencing population fitness and other parameters.
Lipoprotein-associated phospholipase A2 (Lp-PLA2)/platelet-activating factor acetylhydrolase (PAF-AH) has been implicated in the pathogenesis of cardiovascular disease. A therapeutic targeting of this enzyme was challenged by the concern that increased circulating platelet activating factor (PAF) may predispose to or increase the severity of the allergic airway response. The aim of this study was to investigate whether Lp-PLA2 gene deficiency increases the risk of PAF and IgE-mediated inflammatory responses in vitro and in vivo using mouse models.
Lp-PLA2-/- mice were generated and back crossed to the C57BL/6 background. PAF-AH activity was measured using a hydrolysis assay in serum and bronchoalveolar lavage (BAL) samples obtained from mice. Aspergillus fumigatus (Af)-specific serum was prepared for passive allergic sensitization of mice in vivo and mast cells in vitro. β- hexosaminidase release was studied in bone marrow derived mast cells sensitized with Af-specific serum or DNP-IgE and challenged with Af or DNP, respectively. Mice were treated with lipopolysaccharide (LPS) and PAF intratracheally and studied 24 hours later. Mice were sensitized either passively or actively against Af and were studied 48 hours after a single intranasal Af challenge. Airway responsiveness to methacholine, inflammatory cell influx in the lung tissue and BAL, immunoglobulin (ELISA) and cytokine (Luminex) profiles were compared between the wild type (WT) and Lp-PLA2-/- mice.
PAF-AH activity was reduced but not completely abolished in Lp-PLA2-/- serum or by in vitro treatment of serum samples with a high saturating concentration of the selective Lp-PLA2 inhibitor, SB-435495. PAF inhalation significantly enhanced airway inflammation of LPS treated WT and Lp-PLA2-/- mice to a similar extent. Sensitized WT and Lp-PLA2-/- bone-marrow derived mast cells released β-hexosaminidase following stimulation by allergen or IgE crosslinking to equivalent levels. Wild type and Lp-PLA2-/- mice responded to passive or active allergic sensitization by significant IgE production, airway inflammation and hyperresponsiveness after Af challenge. BAL cell influx was not different between these strains while IL-4, IL-5, IL-6 and eotaxin release was attenuated in Lp-PLA2-/- mice. There were no differences in the amount of total IgE levels in the Af sensitized WT and Lp-PLA2-/- mice.
We conclude that Lp-PLA2 deficiency in C57BL/6 mice did not result in a heightened airway inflammation or hyperresponsiveness after PAF/LPS treatment or passive or active allergic sensitization and challenge.
Lp-PLA2; PAF-AH; Knock-out mice; Airway inflammation; IgE; Mast cells; Degranulation
We describe magic-angle spinning NMR experiments designed to elucidate the interstrand architecture of amyloid fibrils. Three methods are introduced for this purpose, two being based on the analysis of long-range 13C-13C correlation spectra and a third based on the identification of intermolecular interactions in 13C-15N spectra. We show, in studies of fibrils formed by the 86-residue SH3 domain of PI3 kinase (PI3-SH3), that efficient 13C-13C correlation spectra display a resonance degeneracy that establishes a parallel, in-register alignment of the proteins in the amyloid fibrils. In addition, this degeneracy can be circumvented to yield direct intermolecular constraints. The 13C-13C experiments are corroborated by 15N-13C correlation spectrum obtained from a mixed [15N,12C]/[14N,13C] sample which directly quantifies interstrand distances. Furthermore, when the spectra are recorded with signal enhancement provided by dynamic nuclear polarization (DNP) at 100 K, we demonstrate a dramatic increase (from 23 to 52) in the number of intermolecular 15N-13C constraints present in the spectra. The increase in the information content is due to the enhanced signal intensities and to the fact that dynamic processes, leading to spectral intensity losses, are quenched at low temperatures. Thus, acquisition of low temperature spectra addresses a problem that is frequently encountered in MAS spectra of proteins. In total the experiments provide 111 intermolecular 13C-13C and 15N-13C constraints that establish that the PI3-SH3 protein strands are aligned in a parallel, in-register arrangement within the amyloid fibril.
Amyloid fibrils; magic angle spinning; dynamic nuclear polarization; phosphatidylinositol-3-kinase SH3 domain (PI3-SH3)
The immunosuppressive drugs used to prevent the rejection of transplanted organs have a narrow therapeutic index. Under treatment results in episodes of rejection leading to either damage or loss of the organ. Over immunosuppression increases the risk of infection and malignancy as well as drug specific complications including diabetes mellitus and nephrotoxicity. There is wide variation in the drug dose required to achieve target blood concentrations and there is often dissociation between pharmacokinetics and pharmacodynamics. Currently, immunosuppressive drug treatment is individualized based on a clinical assessment of the risk of rejection or toxicity. Therapeutic drug monitoring is routinely employed for several immunosuppressive drugs. Pharmacogenetics has the potential to complement therapeutic drug monitoring but clinical benefit has yet to be demonstrated. Novel biomarker-based approaches to risk stratification and pharmacodynamic monitoring are under development and are ready for clinical trials.
CYP3A5; immunosuppression; pharmacogenetics; transplantation
Peroxisome proliferator-activate receptorα (PPARα) activation has been shown in vitro to increase macrophage cholesterol efflux, the initial step in reverse cholesterol transport (RCT). However, it remains unclear whether PPARα activation promotes macrophage RCT in vivo.
Methods and Results
We demonstrated that a specific potent PPARα agonist GW7647 inhibited atherosclerosis and promoted macrophage RCT in hypercholesterolemic mice expressing the human apoA-I gene. We compared the effect of GW7647 on RCT in human apoA-I transgenic (hA-ITg) mice with wild-type (WT) mice and showed that the PPARα agonist promoted RCT in hA-ITg mice to a much greater extent than in WT mice, indicating that human apoA-I expression is important for PPARα-induced RCT. We further investigated the dependence of the macrophage PPARα-LXR pathway on the promotion of RCT by GW7647. Primary murine macrophages lacking PPARα or LXR abolished the ability of GW7647 to promote RCT in hA-ITg mice. In concert, the PPARα agonist promoted cholesterol efflux and ABCA1/ABCG1 expression in primary macrophages and this was also by the PPARα-LXR pathway.
Our observations demonstrate that a potent PPARα agonist promotes macrophage RCT in vivo in a manner that is enhanced by human apoA-I expression and dependent on both macrophage PPARα and LXR expression.
PPARα; LXR; cholesterol efflux; reverse cholesterol transport; apolipoprotein A-I
PPARγ agonists, used in the treatment of Type 2 diabetes, can raise HDL-cholesterol, therefore could potentially stimulate macrophage-to-feces reverse cholesterol transport (RCT). We aimed to test whether PPARγ activation promotes macrophage RCT in vivo. Macrophage RCT was assessed in mice using cholesterol loaded/3H-cholesterol labeled macrophages. PPARγ agonist GW7845 (20 mg/kg/day) did not change 3H-tracer plasma appearance, but surprisingly decreased fecal 3H-free sterol excretion by 43% (P < 0.01) over 48 h. Total free cholesterol efflux from macrophages to serum (collected from control and GW7845 groups) was not different, although ABCA1-mediated efflux was significantly higher with GW7845. To determine the effect of PPARγ activation on HDL cholesterol uptake by different tissues, the metabolic fate of HDL labeled with 3H-cholesteryl ether (CE) was also measured. We observed two-fold increase in HDL derived 3H-CE uptake by adipose tissue (P < 0.005) with concomitant 22% decrease in HDL derived 3H-CE uptake by the liver (P < 0.05) in GW7845 treated wild type mice. This was associated with a significant increase in SR-BI protein expression in adipose tissue, but not liver. The same experiment in SR-BI knockout mice, showed no difference in HDL derived 3H-CE uptake by adipose tissue or liver. In conclusion, PPARγ activation decreases the fecal excretion of macrophage derived cholesterol in mice. This is not due to inhibition of cholesterol efflux from macrophages, but rather involves redirection of effluxed cholesterol from liver towards adipose tissue uptake via SR-BI. This represents a novel mechanism for regulation of RCT and may extend the therapeutic implications of these ligands.
PPARγ; HDL; Reverse cholesterol transport; Adipose tissue; SR-BI
Amyloid fibrils are structurally ordered aggregates of proteins whose formation is associated with many neurodegenerative and other diseases. For that reason, their high resolution structures are of considerable interest and have been studied using a wide range of techniques, notably electron microscopy, x-ray diffraction, and magic angle spinning (MAS) NMR. Because of the excellent resolution in the spectra, MAS NMR is uniquely capable of delivering site-specific, atomic resolution information about all levels of amyloid structure: (1) the monomer, which packs into several (2) protofilaments that in turn associate to form a (3) fibril. Building upon our high resolution structure of the monomer of an amyloid-forming peptide from transthyretin (TTR105-115), we introduce single 1-13C labeled amino acids at seven different sites in the peptide and measure intermolecular carbonyl-carbonyl distances with an accuracy of ~0.11 A. Our results conclusively establish a parallel, in register, topology for the packing of this peptide into a β-sheet and provide constraints essential for the determination of an atomic resolution structure of the fibril. Furthermore, the approach we employ, based on a combination of a double-quantum filtered variant of the DRAWS recoupling sequence and multispin numerical simulations in SPINEVOLUTION, is general and should be applicable to a wide range of systems.
Magic angle spinning; recoupling; transthyretin; protofilament; misfolding
The SH3 domain of the PI3 kinase (PI3-SH3 or PI3K-SH3) readily aggregates into fibrils in vitro and has served as an important model system to investigate the molecular properties and mechanism of formation of amyloid fibrils. We describe the molecular conformation of PI3-SH3 in amyloid fibril form as revealed by magic-angle spinning (MAS) solid-state nuclear magnetic resonance (NMR) spectroscopy. The MAS NMR spectra of these fibrils display excellent resolution, with narrow 13C and 15N line widths, representing a high degree of structural order and the absence of extensive molecular motion for the majority of the polypeptide chain. We have identified the spin-systems of 82 of the 86 residues in the protein, and obtained sequential resonance assignments for 75 of them. Chemical shift analysis indicates that the protein subunits making up the fibril adopt a compact conformation consisting of four well-defined β-sheet regions and four random-coil elements with varying degrees of local dynamics or disorder. The backbone conformation of PI3-SH3 in fibril form differs significantly from that of the native state of the protein, both in secondary structure and in the location of dynamic or disordered segments. The site-specific MAS NMR analysis of PI3-SH3 fibrils we report here is compared with previously published mechanistic and structural data, resulting in a detailed interpretation of the factors that mediate fibril formation by PI3-SH3 and allowing us to propose a possible model of the core structure of the fibrils. Our results confirm the structural similarities between PI3-SH3 fibrils and amyloids directly related to degenerative or infectious diseases.
Late Pleistocene North America hosted at least two divergent and ecologically distinct species of mammoth: the periglacial woolly mammoth (Mammuthus primigenius) and the subglacial Columbian mammoth (Mammuthus columbi). To date, mammoth genetic research has been entirely restricted to woolly mammoths, rendering their genetic evolution difficult to contextualize within broader Pleistocene paleoecology and biogeography. Here, we take an interspecific approach to clarifying mammoth phylogeny by targeting Columbian mammoth remains for mitogenomic sequencing.
We sequenced the first complete mitochondrial genome of a classic Columbian mammoth, as well as the first complete mitochondrial genome of a North American woolly mammoth. Somewhat contrary to conventional paleontological models, which posit that the two species were highly divergent, the M. columbi mitogenome we obtained falls securely within a subclade of endemic North American M. primigenius.
Though limited, our data suggest that the two species interbred at some point in their evolutionary histories. One potential explanation is that woolly mammoth haplotypes entered Columbian mammoth populations via introgression at subglacial ecotones, a scenario with compelling parallels in extant elephants and consistent with certain regional paleontological observations. This highlights the need for multi-genomic data to sufficiently characterize mammoth evolutionary history. Our results demonstrate that the use of next-generation sequencing technologies holds promise in obtaining such data, even from non-cave, non-permafrost Pleistocene depositional contexts.
Parkinson's disease (PD) is traditionally viewed as a motor disorder with a characteristic triad of tremor, rigidity and bradykinesia. There is now increasing awareness that PD is a complex systemic disorder with many nonmotor symptoms (NMS) which include autonomic dysfunction, sleep disorders, sensory and neuropsychiatric features. NMS become more common in severity and frequency with advancing disease when neuropsychiatric features such as cognitive impairment and psychosis dominate the clinical picture. NMS are strongly correlated with quality of life for patients and their families as well as institutional care placement. Despite their importance, NMS are poorly recognized by clinicians and often undeclared by patients. Use of a validated screening tool NMSQuest followed by specific symptom assessment instruments strengthens the recognition and holistic management of NMS in PD. Some NMS such as mood disturbance, anxiety, pain and insomnia may be improved by optimization of dopaminergic therapy. Conversely, psychosis, excess daytime somnolence or impulse control disorder (ICD) may be triggered by dopaminergic drugs. Other NMS such as dementia and severe depression may be unresponsive to dopaminergic treatment and may reflect perturbations in cholinergic, serotonergic or noradrenergic neurotransmitter function. These symptoms are more challenging to manage but may be ameliorated to some extent by agents such as acetylcholinesterase inhibitor or antidepressant drugs. This contribution reviews the evidence for the evaluation and management of key NMS in PD (apathy, anxiety, depression, psychosis, dementia, ICD, sleep disturbance, autonomic dysfunction, pain) and highlights the urgent need for both novel therapies and more controlled trials for current therapeutic strategies.
dementia; depression; dopamine agonists; impulse control disorder; levodopa; nonmotor symptoms; Parkinson's disease; psychosis; sleep disorder
We developed a low-cost, high-throughput microbiome profiling method that uses combinatorial sequence tags attached to PCR primers that amplify the rRNA V6 region. Amplified PCR products are sequenced using an Illumina paired-end protocol to generate millions of overlapping reads. Combinatorial sequence tagging can be used to examine hundreds of samples with far fewer primers than is required when sequence tags are incorporated at only a single end. The number of reads generated permitted saturating or near-saturating analysis of samples of the vaginal microbiome. The large number of reads allowed an in-depth analysis of errors, and we found that PCR-induced errors composed the vast majority of non-organism derived species variants, an observation that has significant implications for sequence clustering of similar high-throughput data. We show that the short reads are sufficient to assign organisms to the genus or species level in most cases. We suggest that this method will be useful for the deep sequencing of any short nucleotide region that is taxonomically informative; these include the V3, V5 regions of the bacterial 16S rRNA genes and the eukaryotic V9 region that is gaining popularity for sampling protist diversity.
Increased lipoprotein-associated phospholipase A2 (Lp-PLA2) activity is associated with increased risk of cardiac events, but it is not known whether Lp-PLA2 is a causative agent. Here we show that selective inhibition of Lp-PLA2 with darapladib reduced development of advanced coronary atherosclerosis in diabetic and hypercholesterolemic swine. Darapladib markedly inhibited plasma and lesion Lp-PLA2 activity and reduced lesion lysophosphatidylcholine content. Analysis of coronary gene expression showed that darapladib exerted a general anti-inflammatory action, substantially reducing the expression of 24 genes associated with macrophage and T lymphocyte functioning. Darapladib treatment resulted in a considerable decrease in plaque area and, notably, a markedly reduced necrotic core area and reduced medial destruction, resulting in fewer lesions with an unstable phenotype. These data show that selective inhibition of Lp-PLA2 inhibits progression to advanced coronary atherosclerotic lesions and confirms a crucial role of vascular inflammation independent from hypercholesterolemia in the development of lesions implicated in the pathogenesis of myocardial infarction and stroke.
PPARδ agonism increases HDL-cholesterol and has therefore the potential to stimulate macrophage-to-feces reverse cholesterol transport (RCT). To test whether PPARδ activation promotes RCT in mice, in vivo macrophage RCT was assessed using cholesterol loaded/3H-cholesterol labeled macrophages injected intraperitoneally. PPARδ agonist GW0742 (10mg/kg/day) did not change 3H -tracer plasma appearance, but increased fecal 3H-free sterols excretion by 103% (p<0.005) over 48 hours. Total free cholesterol efflux from macrophages to serum (collected from both control and GW0742 groups) was not different, although ABCA1-mediated efflux was significantly higher with GW0742. The metabolic fate of HDL labeled with 3H-cholesteryl ether or 3H-cholesteryl oleate was also measured. While 3H-cholesteryl ether tissue uptake was unchanged, the 3H-tracer recovered in fecal free sterol fraction after 3H-cholesteryl oleate injection increased by 88% with GW0742 (p<0.0005). This was associated with a lower Niemann Pick C1 like 1 (NPC1L1) mRNA expression in the small intestine (p<0.05). The same experiments in mice treated with ezetimibe, which blocks NPC1L1, showed a similar 2-fold increase in fecal free sterol excretion after labeled macrophages or HDL injection.
In conclusion, PPARδ activation enhances excretion of macrophage or HDL-derived cholesterol in feces through reduced NPC1L1 expression in mice, comparable to the effect of ezetimibe.
intestine; cholesterol; lipoproteins; reverse cholesterol transport
In the fusion pathway of trophoblast differentiation, stem villous cytotrophoblast cells proliferate and daughter cells differentiate and fuse with existing syncytiotrophoblast to maintain the multi-nucleated layer. Integrin-linked kinase (ILK) is highly expressed in 1st and 2nd trimester villous cytotrophoblast cells, yet barely detectable in syncytiotrophoblast, thus we examined the potential role of ILK in aiding trophoblast fusion.
The temporal/spatial expression and activity of ILK were determined in BeWo cells undergoing syncytialization by immunoblot and immunofluorescence analyses. BeWo cells were also transfected with pEGFP expression vectors containing wildtype or two mutant ILK cDNA constructs. The incidence of cell fusion in transfected cells grown under syncytialization conditions was then scored by the presence or absence of E-cadherin immunostaining. Beta-hCG expression in transfected cells, a marker of syncytiotrophoblast hormonal differentiation, was also similarly assessed.
ILK catalytic activity increased and ILK began to increasingly localize to BeWo cell nuclei during syncytialization in correlation with increased pAkt and Snail protein expression. Syncytialization was also significantly elevated (p < 0.05) in BeWo cells expressing constitutively active (ca)-ILK vs cells containing empty vector or dn-ILK. Furthermore, cytoplasmic Beta-hCG expression markedly increased (p < 0.05) in cells expressing wt- and ca-ILK.
ILK-facilitated syncytialization is dependent, at least in part, on ILK catalytic activity while hormonal differentiation appears dependent on both ILK-associated protein interactions and catalytic activity. This study demonstrates that ILK plays a novel role in BeWo syncytialization and differentiation, perhaps through an ILK-Akt-Snail pathway, and implicates ILK in the same process in villous cytotrophoblasts in vivo.
It is now widely accepted that novel infectious disease can be a leading cause of serious population decline and even outright extinction in some invertebrate and vertebrate groups (e.g., amphibians). In the case of mammals, however, there are still no well-corroborated instances of such diseases having caused or significantly contributed to the complete collapse of species. A case in point is the extinction of the endemic Christmas Island rat (Rattus macleari): although it has been argued that its disappearance ca. AD 1900 may have been partly or wholly caused by a pathogenic trypanosome carried by fleas hosted on recently-introduced black rats (Rattus rattus), no decisive evidence for this scenario has ever been adduced. Using ancient DNA methods on samples from museum specimens of these rodents collected during the extinction window (AD 1888–1908), we were able to resolve unambiguously sequence evidence of murid trypanosomes in both endemic and invasive rats. Importantly, endemic rats collected prior to the introduction of black rats were devoid of trypanosome signal. Hybridization between endemic and black rats was also previously hypothesized, but we found no evidence of this in examined specimens, and conclude that hybridization cannot account for the disappearance of the endemic species. This is the first molecular evidence for a pathogen emerging in a naïve mammal species immediately prior to its final collapse.
Myometrial growth and remodeling of the cytoskeleton and focal adhesions during late pregnancy may be critical aspects of myometrial activation and thus labour. Yet our understanding of these aspects is inhibited by the paucity of information concerning the components of focal adhesions in the myometrium. The focal adhesion protein hydrogen peroxide-inducible clone-5 (Hic-5) has recently been found in mononuclear smooth muscle but was not examined in the myometrium during pregnancy. Thus, the goal of this study was to characterize Hic-5 mRNA and protein expression in the rat myometrium during pregnancy and labour.
Rat myometrium samples were obtained from non-pregnant animals, pregnant animals on days (d) 6, 12, 15, 17, 19, 21, 22, 23 (active labour) and 1 day postpartum (PP). In addition, myometrium samples were collected from rats within a progesterone-delayed labour paradigm. Hic-5 mRNA expression was analyzed by Northern blot analysis while Hic-5 protein expression was examined by immunoblot and immunofluorescence analysis.
Hic-5 mRNA expression on d15, d19 and d21 was found to be significantly elevated compared to d6 and d12 of pregnancy and expression on d23 was significantly elevated over d6 (p < 0.05). Immunofluorescence analysis demonstrated that detection of Hic-5 protein in the circular muscle layer appeared to increase from d17 onwards, except PP, and Hic-5 was detectable in the cell cytoplasm and more continuously associated with myometrial cell membranes. In the longitudinal muscle layer Hic-5 was readily detectable by d15 and thereafter and primarily associated at myometrial cell membranes. Co-immunofluorescence analysis of potential Hic-5 and focal adhesion kinase (FAK) association in situ demonstrated a limited level of co-localization on d19, d23 and PP in the circular muscle layer while in the longitudinal muscle layer Hic-5 and FAK were readily co-localized at myometrial cell membranes.
Hic-5 is highly expressed in the rat myometrium during late pregnancy and labour and co-localizes with FAK in situ. Our results are consistent with a potential role for Hic-5 in focal adhesion remodeling in the rat myometrium during late pregnancy.
Nuclear insertions of mitochondrial sequences (NuMts) have been identified in a wide variety of organisms. Trafficking of genetic material from the mitochondria to the nucleus has occurred frequently during mammalian evolution and can lead to the production of a large pool of sequences with varying degrees of homology to organellar mitochondrial DNA (mtDNA) sequences. This presents both opportunities and challenges for forensics, population genetics, evolutionary genetics, conservation biology and the study of DNA from ancient samples. Here we present a case in which difficulties in ascertaining the organellar mtDNA sequence from modern samples hindered their comparison to ancient DNA sequences.
We obtained mitochondrial hypervariable region (HVR) sequences from six ancient samples of tundra muskox (Ovibos moschatus) that were reproducible but distinct from modern muskox sequences reported previously. Using the same PCR primers applied to the ancient specimens and the primers used to generate the modern muskox DNA sequences in a previous study, we failed to definitively identify the organellar sequence from the two modern muskox samples tested. Instead of anticipated sequence homogeneity, we obtained multiple unique sequences from both hair and blood of one modern specimen. Sequencing individual clones of a >1 kb PCR fragment from modern samples did not alleviate the problem as there was not a consistent match across the entire length of the sequences to Ovibos when compared to sequences in GenBank.
In specific taxa, due to nuclear insertions some regions of the mitochondrial genome may not be useful for the characterization of modern or ancient DNA.
The modern wildherd of the tundra muskox (Ovibos moschatus) is native only to the New World (northern North America and Greenland), and its genetic diversity is notably low. However, like several other megafaunal mammals, muskoxen enjoyed a holarctic distribution during the late Pleistocene. To investigate whether collapse in range and loss of diversity might be correlated, we collected mitochondrial sequence data (hypervariable region and cytochrome b) from muskox fossil material recovered from localities in northeastern Asia and the Arctic Archipelago of northern North America, dating from late Pleistocene to late Holocene, and compared our results to existing databases for modern muskoxen.
Two classes of haplotypes were detected in the fossil material. "Surviving haplotypes" (SHs), closely similar or identical to haplotypes found in modern muskoxen and ranging in age from ~22,000 to ~160 yrbp, were found in all New World samples as well as some samples from northeastern Asia. "Extinct haplotypes" (EHs), dating between ~44,000 and ~18,000 yrbp, were found only in material from the Taimyr Peninsula and New Siberian Islands in northeastern Asia. EHs were not found in the Holocene muskoxen specimens available for this study, nor have they been found in other studies of extant muskox populations.
We provisionally interpret this evidence as showing that genetic variability was reduced in muskoxen after the Last Glacial Maximum but before the mid-Holocene, or roughly within the interval 18,000-4,000 yrbp. Narrowing this gap further will require the recovery of more fossils and additional genetic information from this interval.
Endogenous retrovirus-like elements (ERV-Ls, primed with tRNA leucine) are a diverse group of reiterated sequences related to foamy viruses and widely distributed among mammals. As shown in previous investigations, in many primates and rodents this class of elements has remained transpositionally active, as reflected by increased copy number and high sequence diversity within and among taxa.
Here we examine whether proviral-like sequences may be suitable molecular probes for investigating the phylogeny of groups known to have high element diversity. As a test we characterized ERV-Ls occurring in a sample of extant members of superorder Uranotheria (Asian and African elephants, manatees, and hyraxes). The ERV-L complement in this group is even more diverse than previously suspected, and there is sequence evidence for active expansion, particularly in elephantids. Many of the elements characterized have protein coding potential suggestive of activity.
In general, the evidence supports the hypothesis that the complement had a single origin within basal Uranotheria.