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1.  Faunal record identifies Bering isthmus conditions as constraint to end-Pleistocene migration to the New World 
Human colonization of the New World is generally believed to have entailed migrations from Siberia across the Bering isthmus. However, the limited archaeological record of these migrations means that details of the timing, cause and rate remain cryptic. Here, we have used a combination of ancient DNA, 14C dating, hydrogen and oxygen isotopes, and collagen sequencing to explore the colonization history of one of the few other large mammals to have successfully migrated into the Americas at this time: the North American elk (Cervus elaphus canadensis), also known as wapiti. We identify a long-term occupation of northeast Siberia, far beyond the species’s current Old World distribution. Migration into North America occurred at the end of the last glaciation, while the northeast Siberian source population became extinct only within the last 500 years. This finding is congruent with a similar proposed delay in human colonization, inferred from modern human mitochondrial DNA, and suggestions that the Bering isthmus was not traversable during parts of the Late Pleistocene. Our data imply a fundamental constraint in crossing Beringia, placing limits on the age and mode of human settlement in the Americas, and further establish the utility of ancient DNA in palaeontological investigations of species histories.
doi:10.1098/rspb.2013.2167
PMCID: PMC3871309  PMID: 24335981
ancient DNA; Beringia; Bering isthmus; Pleistocene; wapiti
2.  Preferential vulnerability of astroglia and glial precursors to combined opioid and HIV-1 Tat exposure in vitro 
The European journal of neuroscience  2004;19(12):3171-3182.
Human immunodeficiency virus-1 (HIV) infection can cause characteristic neural defects such as progressive motor dysfunction, striatal pathology, and gliosis. Recent evidence suggests that HIV-induced pathogenesis is exacerbated by heroin abuse and that the synergistic neurotoxicity is a direct effect of heroin on the CNS, an alarming observation considering the high incidence of HIV infection with injection drug abuse. Although HIV infection results in neurodegeneration, neurons themselves are not directly infected. Instead, HIV affects microglia and astroglia, which subsequently contributes to the neurodegenerative changes. Opioid receptors are widely expressed by macroglia and macroglial precursors, and the activation of µ-opioid receptors can modulate programmed cell death, as well as the response of neural cells to cytotoxic insults. For this reason, we questioned whether opioid drugs might modify the vulnerability of macroglia and macroglial precursors to HIV-1 Tat protein. To address this problem, the effects of morphine and/or HIV Tat1–72 on the viability of macroglia and macroglial precursors were assessed in mixed-glial cultures derived from mouse striatum. Our findings indicate that sustained exposure to morphine and Tat1–72 viral protein induces the preferential death of glial precursors and immature oligodendroglia. Moreover, the increased cell death is mediated by µ-opioid receptors and accompanied by the activation of caspase-3. Our results imply that opiates can enhance the cytotoxicity of HIV-1 Tat through direct actions on glial precursors and/or astroglia, suggesting novel cellular targets for HIV-opiate interactions.
doi:10.1111/j.0953-816X.2004.03461.x
PMCID: PMC4305445  PMID: 15217373
Human immunodeficiency virus; opioid receptors; striatum; drug abuse; morphine; glial precursors
3.  Argon Inhalation Attenuates Retinal Apoptosis after Ischemia/Reperfusion Injury in a Time- and Dose-Dependent Manner in Rats 
PLoS ONE  2014;9(12):e115984.
Purpose
Retinal ischemia and reperfusion injuries (IRI) permanently affect neuronal tissue and function by apoptosis and inflammation due to the limited regenerative potential of neurons. Recently, evidence emerged that the noble gas Argon exerts protective properties, while lacking any detrimental or adverse effects. We hypothesized that Argon inhalation after IRI would exert antiapoptotic effects in the retina, thereby protecting retinal ganglion cells (RGC) of the rat's eye.
Methods
IRI was performed on the left eyes of rats (n = 8) with or without inhaled Argon postconditioning (25, 50 and 75 Vol%) for 1 hour immediately or delayed after ischemia (i.e. 1.5 and 3 hours). Retinal tissue was harvested after 24 hours to analyze mRNA and protein expression of Bcl-2, Bax and Caspase-3, NF-κB. Densities of fluorogold-prelabeled RGCs were analyzed 7 days after injury in whole-mounts. Histological tissue samples were prepared for immunohistochemistry and blood was analyzed regarding systemic effects of Argon or IRI. Statistics were performed using One-Way ANOVA.
Results
IRI induced RGC loss was reduced by Argon 75 Vol% inhalation and was dose-dependently attenuated by lower concentrations, or by delayed Argon inhalation (1504±300 vs. 2761±257; p<0.001). Moreover, Argon inhibited Bax and Bcl-2 mRNA expression significantly (Bax: 1.64±0.30 vs. 0.78±0.29 and Bcl-2: 2.07±0.29 vs. 0.99±0.22; both p<0.01), as well as caspase-3 cleavage (1.91±0.46 vs. 1.05±0.36; p<0.001). Expression of NF-κB was attenuated significantly. Immunohistochemistry revealed an affection of Müller cells and astrocytes. In addition, IRI induced leukocytosis was reduced significantly after Argon inhalation at 75 Vol%.
Conclusion
Immediate and delayed Argon postconditioning protects IRI induced apoptotic loss of RGC in a time- and dose-dependent manner, possibly mediated by the inhibition of NF-κB. Further studies need to evaluate Argon's possible role as a therapeutic option.
doi:10.1371/journal.pone.0115984
PMCID: PMC4275290  PMID: 25535961
4.  Evaluating Population Receptive Field Estimation Frameworks in Terms of Robustness and Reproducibility 
PLoS ONE  2014;9(12):e114054.
Within vision research retinotopic mapping and the more general receptive field estimation approach constitute not only an active field of research in itself but also underlie a plethora of interesting applications. This necessitates not only good estimation of population receptive fields (pRFs) but also that these receptive fields are consistent across time rather than dynamically changing. It is therefore of interest to maximize the accuracy with which population receptive fields can be estimated in a functional magnetic resonance imaging (fMRI) setting. This, in turn, requires an adequate estimation framework providing the data for population receptive field mapping. More specifically, adequate decisions with regard to stimulus choice and mode of presentation need to be made. Additionally, it needs to be evaluated whether the stimulation protocol should entail mean luminance periods and whether it is advantageous to average the blood oxygenation level dependent (BOLD) signal across stimulus cycles or not. By systematically studying the effects of these decisions on pRF estimates in an empirical as well as simulation setting we come to the conclusion that a bar stimulus presented at random positions and interspersed with mean luminance periods is generally most favorable. Finally, using this optimal estimation framework we furthermore tested the assumption of temporal consistency of population receptive fields. We show that the estimation of pRFs from two temporally separated sessions leads to highly similar pRF parameters.
doi:10.1371/journal.pone.0114054
PMCID: PMC4252088  PMID: 25463652
5.  Emotion unfolded by motion: a role for parietal lobe in decoding dynamic facial expressions 
Facial expressions convey important emotional and social information and are frequently applied in investigations of human affective processing. Dynamic faces may provide higher ecological validity to examine perceptual and cognitive processing of facial expressions. Higher order processing of emotional faces was addressed by varying the task and virtual face models systematically. Blood oxygenation level-dependent activation was assessed using functional magnetic resonance imaging in 20 healthy volunteers while viewing and evaluating either emotion or gender intensity of dynamic face stimuli. A general linear model analysis revealed that high valence activated a network of motion-responsive areas, indicating that visual motion areas support perceptual coding for the motion-based intensity of facial expressions. The comparison of emotion with gender discrimination task revealed increased activation of inferior parietal lobule, which highlights the involvement of parietal areas in processing of high level features of faces. Dynamic emotional stimuli may help to emphasize functions of the hypothesized ‘extended’ over the ‘core’ system for face processing.
doi:10.1093/scan/nss092
PMCID: PMC3831559  PMID: 22962061
dynamic facial expressions; emotion perception; emotion–cognition interaction; inferior parietal lobule
6.  Low-dose intravenous immunoglobulin treatment for complex regional pain syndrome (LIPS): study protocol for a randomized controlled trial 
Trials  2014;15(1):404.
Background
Longstanding complex regional pain syndrome (CRPS) is refractory to treatment with established analgesic drugs in most cases, and for many patients, alternative pain treatment approaches, such as with neuromodulation devices or rehabilitation methods, also do not work. The development of novel, effective treatment technologies is, therefore, important. There are preliminary data suggesting that low-dose immunoglobulin treatment may significantly reduce pain from longstanding CRPS.
Methods/Design
LIPS is a multicentre (United Kingdom), double-blind, randomised parallel group, placebo-controlled trial, designed to evaluate the efficacy, safety, and tolerability of intravenous immunoglobulin (IVIg) 0.5 g/kg plus standard treatment, versus matched placebo plus standard treatment in 108 patients with longstanding complex regional pain syndrome. Participants with moderate or severe CRPS of between 1 and 5 years duration will be randomly allocated to receive IVIg 0.5 g/kg (IntratectTM 50 g/l solution for infusion) or matching placebo administered day 1 and day 22 after randomisation, followed by two optional doses of open-label medication on day 43 after randomisation and on day 64 after randomisation. The primary outcome is the patients’ pain intensity in the IVIG group compared with the placebo group, between 6 and 42 days after randomisation. The primary trial objective is to confirm the efficacy and confidently determine the effect size of the IVIG treatment technology in this group of patients.
Trial registration
ISRCTN42179756 (Registered 28 June 13).
Electronic supplementary material
The online version of this article (doi:10.1186/1745-6215-15-404) contains supplementary material, which is available to authorized users.
doi:10.1186/1745-6215-15-404
PMCID: PMC4226877  PMID: 25344328
CRPS; immunoglobulin; IVIg; pain
8.  Quantitative Phosphoproteomics of Cytotoxic T Cells to Reveal Protein Kinase D 2 Regulated Networks* 
Molecular & Cellular Proteomics : MCP  2014;13(12):3544-3557.
The focus of the present study was to characterize the phosphoproteome of cytotoxic T cells and to explore the role of the serine threonine kinase PKD2 (Protein Kinase D2) in the phosphorylation networks of this key lymphocyte population. We used Stable Isotope Labeling of Amino acids in Culture (SILAC) combined with phosphopeptide enrichment and quantitative mass-spectrometry to determine the impact of PKD2 loss on the cytotoxic T cells phosphoproteome. We identified 15,871 phosphorylations on 3505 proteins in cytotoxic T cells. 450 phosphosites on 281 proteins were down-regulated and 300 phosphosites on 196 proteins were up-regulated in PKD2 null cytotoxic T cells. These data give valuable new insights about the protein phosphorylation networks operational in effector T cells and reveal that PKD2 regulates directly and indirectly about 5% of the cytotoxic T-cell phosphoproteome. PKD2 candidate substrates identified in this study include proteins involved in two distinct biological functions: regulation of protein sorting and intracellular vesicle trafficking, and control of chromatin structure, transcription, and translation. In other cell types, PKD substrates include class II histone deacetylases such as HDAC7 and actin regulatory proteins such as Slingshot. The current data show these are not PKD substrates in primary T cells revealing that the functional role of PKD isoforms is different in different cell lineages.
doi:10.1074/mcp.M113.037242
PMCID: PMC4256504  PMID: 25266776
9.  Hyperacute Rejection of a Living Unrelated Kidney Graft 
Case Reports in Medicine  2014;2014:613641.
We present a case report of a 59-year-old man, who received a blood group identical living unrelated kidney graft. This was his second kidney transplantation. Pretransplant T-cell crossmatch resulted negative. B-cell crossmatch, which is not considered a strict contraindication for transplantation, resulted positive. During surgery no abnormalities occurred. Four hours after the transplantation diuresis suddenly decreased. In an immediately performed relaparotomy the transplanted kidney showed signs of hyperacute rejection and had to be removed. Pathological examination was consistent with hyperacute rejection. Depositions of IgM or IgG antibodies were not present in pathologic evaluation of the rejected kidney, suggesting that no irregular endothelial specific antibodies had been involved in the rejection. We recommend examining more closely recipients of second allografts, considering not only a positive T-cell crossmatch but also a positive B-cell crossmatch as exclusion criteria for transplantation.
doi:10.1155/2014/613641
PMCID: PMC4182006  PMID: 25317177
10.  Links between Belowground and Aboveground Resource-Related Traits Reveal Species Growth Strategies that Promote Invasive Advantages 
PLoS ONE  2014;9(8):e104189.
Belowground processes are rarely considered in comparison studies of native verses invasive species. We examined relationships between belowground fine root production and lifespan, leaf phenology, and seasonal nitrogen dynamics of Lonicera japonica (non-native) versus L. sempervirens (native) and Frangula alnus (non-native) versus Rhamnus alnifolia (native), over time. First and second order fine roots were monitored from 2010 to 2012 using minirhizotron technology and rhizotron windows. 15N uptake of fine roots was measured across spring and fall seasons. Significant differences in fine root production across seasons were seen between Lonicera species, but not between Frangula and Rhamnus, with both groups having notable asynchrony in regards to the timing of leaf production. Root order and the number of root neighbors at the time of root death were the strongest predictors of root lifespan of both species pairs. Seasonal 15N uptake was higher in spring than in the fall, which did not support the need for higher root activity to correspond with extended leaf phenology. We found higher spring 15N uptake in non-native L. japonica compared to native L. sempervirens, although there was no difference in 15N uptake between Frangula and Rhamnus species. Our findings indicate the potential for fast-growing non-native Lonicera japonica and Frangula alnus to outcompete native counterparts through differences in biomass allocation, root turnover, and nitrogen uptake, however evidence that this is a general strategy of invader dominance is limited.
doi:10.1371/journal.pone.0104189
PMCID: PMC4126695  PMID: 25105975
11.  De Novo Prion Aggregates Trigger Autophagy in Skeletal Muscle 
Journal of Virology  2014;88(4):2071-2082.
ABSTRACT
In certain sporadic, familial, and infectious prion diseases, the prion protein misfolds and aggregates in skeletal muscle in addition to the brain and spinal cord. In myocytes, prion aggregates accumulate intracellularly, yet little is known about clearance pathways. Here we investigated the clearance of prion aggregates in muscle of transgenic mice that develop prion disease de novo. In addition to neurodegeneration, aged mice developed a degenerative myopathy, with scattered myocytes containing ubiquitinated, intracellular prion inclusions that were adjacent to myocytes lacking inclusions. Myocytes also showed elevated levels of the endoplasmic reticulum chaperone Grp78/BiP, suggestive of impaired protein degradation and endoplasmic reticulum stress. Additionally, autophagy was induced, as indicated by increased levels of beclin-1 and LC3-II. In C2C12 myoblasts, inhibition of autophagosome maturation or lysosomal degradation led to enhanced prion aggregation, consistent with a role for autophagy in prion aggregate clearance. Taken together, these findings suggest that the induction of autophagy may be a central strategy for prion aggregate clearance in myocytes.
IMPORTANCE
doi:10.1128/JVI.02279-13
PMCID: PMC3911572  PMID: 24307586
12.  BK Viremia Precedes Hemorrhagic Cystitis in Children Undergoing Allogeneic Hematopoietic Stem Cell Transplantation 
BK virus is associated with hemorrhagic cystitis after hematopoietic stem cell transplantation (HSCT), although evidence supporting a causal relationship remains limited. Although BK viruria is common after HSCT, BK viremia may better predict clinically significant cystitis, similar to its predictive value for nephropathy after kidney transplantation. We hypothesized that BK viremia would precede hemorrhagic cystitis in a cohort of 88 consecutive children prospectively enrolled to originally study thrombotic microangiopathy in the first 100 days after allogeneic HSCT. Cox regression models with time-varying covariates assessed the association between different BK viremia cutoffs and the development of hemorrhagic cystitis, defined as at least macroscopic hematuria. Subjects with a peak plasma BK viral load 1 to 9999 copies/mL had an adjusted hazard ratio of 4.2 (95% confidence interval (CI), 1.3 to 13.7) for the development of hemorrhagic cystitis. Those with peak BK viremia >100,000 copies/mL had an adjusted hazard ratio of 116.8 (95% CI, 12 to 1136) for cystitis. Other independent risk factors for hemorrhagic cystitis included age >7 years and HHV-6 viremia. Neither graft-versus-host disease nor achieving engraftment increased the risk for cystitis. If therapeutic strategies are found to be effective, these observations may support screening for BK viremia after HSCT, as currently recommended for other DNA viruses.
doi:10.1016/j.bbmt.2013.05.002
PMCID: PMC3774139  PMID: 23665115
BK virus; Hemorrhagic cystitis; Transplantation; Pediatrics
13.  The lymphoid follicle variant of dermatomyositis 
Objective:
To investigate the clinical and morphologic spectrum of early adult–onset dermatomyositis (DM), an inflammatory disease that affects small vessels of the muscle and the skin.
Methods:
Histologic evaluation of frozen muscle samples was employed to visualize the cellular organization of ectopic lymphoid structures in muscle biopsies obtained from 2 patients diagnosed with DM. Clinical presentation and morphologic features, as well as treatment and follow-up, were assessed and documented. Electron microscopy was used to confirm the light microscopic diagnosis of DM. Clonality analysis of B-cell populations using PCR was performed.
Results:
Muscle biopsy of both patients fulfilled the morphologic European Neuromuscular Centre criteria of DM. Analyses of muscle biopsy samples revealed ectopic lymphoid follicle-like structures that showed a remarkable similarity to secondary lymphoid organs (SLOs) with distinct T- and B-cell compartmentalization. Our 2 patients exhibited an atypical and mild clinical presentation and responded favorably to therapy.
Conclusions:
The clinical and histopathologic features of DM can be atypical, and the presence of SLOs is not inevitably linked to an unfavorable prognosis.
doi:10.1212/NXI.0000000000000019
PMCID: PMC4202675  PMID: 25340071
14.  Role of Intravitreal Antivascular Endothelial Growth Factor Injections for Choroidal Neovascularization due to Choroidal Osteoma 
Journal of Ophthalmology  2014;2014:210458.
We treated 26 eyes of 25 young patients having a mean age of 30 years with intravitreal vascular endothelial growth factor (VEGF) inhibitor for choroidal new vessel (CNV) formation overlying choroidal osteoma over a mean follow-up of 26 months. Mean number of injections was 2.4 at 6 months, 3.2 at 12 months, and 5.5 at 24 months. CNV was subfoveal in 14 eyes, juxtafoveal in 5, extrafoveal in 5, and peripapillary in 2. By paired comparison, mean decrease from baseline was 119.7 microns at 6 months (n = 15; P = 0.001), 105.3 microns at 1 year (n = 10; P = 0.03), and 157.6 microns at 2 years (n = 7; P = 0.08). BCVA improved by 3.3 lines at 6 months after therapy (n = 26; P < 0.001), 2.8 lines (n = 20; P = 0.01) at 1 year, and 3.1 lines (n = 13; P = 0.049) at 2 years. We conclude that intravitreal anti-VEGF injections improve vision in majority of eyes with CNV from choroidal osteoma.
doi:10.1155/2014/210458
PMCID: PMC4132478  PMID: 25147732
15.  The Synthesis and Pharmacological Evaluation of Adamantane-Derived Indoles: Cannabimimetic Drugs of Abuse 
ACS Chemical Neuroscience  2013;4(7):1081-1092.
Two novel adamantane derivatives, adamantan-1-yl(1-pentyl-1H-indol-3-yl)methanone (AB-001) and N-(adamtan-1-yl)-1-pentyl-1H-indole-3-carboxamide (SDB-001), were recently identified as cannabimimetic indoles of abuse. Conflicting anecdotal reports of the psychoactivity of AB-001 in humans, and a complete dearth of information about the bioactivity of SDB-001, prompted the preparation of AB-001, SDB-001, and several analogues intended to explore preliminary structure–activity relationships within this class. This study sought to elucidate which structural features of AB-001, SDB-001, and their analogues govern the cannabimimetic potency of these chemotypes in vitro and in vivo. All compounds showed similar full agonist profiles at CB1 (EC50 = 16–43 nM) and CB2 (EC50 = 29–216 nM) receptors in vitro using a FLIPR membrane potential assay, with the exception of SDB-002, which demonstrated partial agonist activity at CB2 receptors. The activity of AB-001, AB-002, and SDB-001 in rats was compared to that of Δ9-tetrahydrocannabinol (Δ9-THC) and cannabimimetic indole JWH-018 using biotelemetry. SDB-001 dose-dependently induced hypothermia and reduced heart rate (maximal dose 10 mg/kg) with potency comparable to that of Δ9-tetrahydrocannabinol (Δ9-THC, maximal dose 10 mg/kg), and lower than that of JWH-018 (maximal dose 3 mg/kg). Additionally, the changes in body temperature and heart rate affected by SDB-001 are of longer duration than those of Δ9-THC or JWH-018, suggesting a different pharmacokinetic profile. In contrast, AB-001, and its homologue, AB-002, did not produce significant hypothermic and bradycardic effects, even at relatively higher doses (up to 30 mg/kg), indicating greatly reduced potency compared to Δ9-THC, JWH-018, and SDB-001.
doi:10.1021/cn400035r
PMCID: PMC3715837  PMID: 23551277
indole; AB-001; Δ9-tetrahydrocannabinol; cannabinoid receptor; drug abuse
16.  Structural and Functional Characterization of MppR, an Enduracididine Biosynthetic Enzyme from Streptomyces hygroscopicus: Functional Diversity in the Acetoacetate Decarboxylase-Like Superfamily 
Biochemistry  2013;52(26):4492-4506.
The non-proteinogenic amino acid enduracididine is a critical component of the mannopeptimycins, cyclic glycopeptide antibiotics with activity against drug-resistant pathogens including methicillin-resistant Staphylococcus aureus. Enduracididine is produced in Streptomyces hygroscopicus by three enzymes, MppP, MppQ, and MppR. Based on primary sequence analysis, MppP and Q are pyridoxal-5'-phosphate-dependent aminotransferases; MppR shares low, but significant, sequence identity with acetoacetate decarboxylase. The exact reactions catalyzed by each enzyme, and the intermediates involved in the route to enduracididine are currently unknown. Herein we present biochemical and structural characterization of MppR that demonstrates a catalytic activity for this enzyme and provides clues about its role in enduracididine biosynthesis. Bioinformatic analysis shows that MppR belongs to a previously uncharacterized family within the acetoacetate decarboxylase-like superfamily (ADCSF) and suggests that MppR-like enzymes may catalyze reactions diverging from the well-characterized, prototypical ADCSF decarboxylase activity. MppR shares a high degree of structural similarity with acetoacetate decarboxylase, though the respective quaternary structures differ markedly and structural differences in the active site explain the observed loss of decarboxylase activity. The crystal structure of MppR in the presence of a mixture of pyruvate and 4-imidazolecarboxaldehyde shows that MppR catalyzes the aldol condensation of these compounds and subsequent dehydration. Surprisingly, the structure of MppR in the presence of a mixture of "4-hydroxy-2-ketoarginine" and "2-ketoenduracididine" shows only the correct 4R-enantiomer of "2-ketoenduracididine" bound to the enzyme. These data, together with bioinformatic analysis of MppR homologs, identifies a novel family within the acetoacetate decarboxylase-like superfamily with divergent active site structure and, consequently, biochemical function.
doi:10.1021/bi400397k
PMCID: PMC3743547  PMID: 23758195
Nonribosomal peptide antibiotics; mannopeptimycin; acetoacetate decarboxylase; pyruvate aldolase
17.  Nesfatin-1: a novel inhibitory regulator of food intake and body weight 
Summary
The protein nucleobindin 2 (NUCB2) or NEFA (DNA binding/EF-hand/acidic amino acid rich region) was identified over a decade ago and implicated in intracellular processes. New developments came with the report that post-translational processing of hypothalamic NUCB2 may result in nesfatin-1, nesfatin-2 and nesfatin-3 and convergent studies showing that nesfatin-1 and full length NUCB2 injected in the brain potently inhibit the dark phase food intake in rodents including leptin receptor deficient Zucker rats. Nesfatin-1 also reduces body weight gain, suggesting a role as a new anorexigenic factor and modulator of energy balance. In light of the obesity epidemic and its associated diseases, underlying new mechanisms regulating food intake may be promising targets in the drug treatment of obese patients particularly as the vast majority of them display reduced leptin sensitivity or leptin resistance while nesfatin-1’s mechanism of action is leptin independent. Although much progress on the localization of NUCB2/nesfatin-1 in the brain and periphery as well as on the understanding of nesfatin-1’s anorexic effect have been achieved during the past three years, several important mechanisms have yet to be unraveled such as the identification of the nesfatin-1 receptor and the regulation of NUCB2 processing and nesfatin-1 release.
doi:10.1111/j.1467-789X.2010.00770.x
PMCID: PMC4079085  PMID: 20546141
Food intake; hypothalamus; nesfatin-1/NUCB2; X/A-like cells
18.  Association of TNFRSF10D DNA-Methylation with the Survival of Melanoma Patients 
In this retrospective pilot study, the DNA-methylation status of genes that have been demonstrated to be involved in melanoma carcinogenesis was analyzed in order to identify novel biomarkers for the risk assessment of melanoma patients. We analyzed DNA extracted from punch-biopsies from 68 formalin-fixed paraffin-embedded (FFPE) melanoma specimens. Using MethyLight PCR, we examined 20 genes in specimens from a training set comprising 36 melanoma patients. Selected candidate genes were validated in a test set using FFPE tissue samples from 32 melanoma patients. First, we identified the TNFRSF10D DNA-methylation status (TNFRSF10D methylated vs. unmethylated) as a prognostic marker for overall (p = 0.001) and for relapse-free survival (p = 0.008) in the training set. This finding was confirmed in the independent test set (n = 32; overall survival p = 0.041; relapse-free survival p = 0.012). In a multivariate Cox-regression analysis including all patients, the TNFRSF10D DNA-methylation status remained as the most significant prognostic parameter for overall and relapse-free survival (relative-risk (RR) of death, 4.6 (95% CI: 2.0–11.0; p < 0.001), RR of relapse, 7.2 (95% CI: 2.8–18.3; p < 0.001)). In this study, we demonstrate that TNFRSF10D DNA-methylation analysis of a small tissue-punch from archival FFPE melanoma tissue is a promising approach to provide prognostic information in patients with melanoma.
doi:10.3390/ijms150711984
PMCID: PMC4139825  PMID: 25003639
cancer biomarker; epigenomics; DNA-methylation; prognosis; translational cancer research
19.  Species-specific responses of Late Quaternary megafauna to climate and humans 
Nature  2011;479(7373):359-364.
Despite decades of research, the roles of climate and humans in driving the dramatic extinctions of large-bodied mammals during the Late Quaternary remain contentious. We use ancient DNA, species distribution models and the human fossil record to elucidate how climate and humans shaped the demographic history of woolly rhinoceros, woolly mammoth, wild horse, reindeer, bison and musk ox. We show that climate has been a major driver of population change over the past 50,000 years. However, each species responds differently to the effects of climatic shifts, habitat redistribution and human encroachment. Although climate change alone can explain the extinction of some species, such as Eurasian musk ox and woolly rhinoceros, a combination of climatic and anthropogenic effects appears to be responsible for the extinction of others, including Eurasian steppe bison and wild horse. We find no genetic signature or any distinctive range dynamics distinguishing extinct from surviving species, underscoring the challenges associated with predicting future responses of extant mammals to climate and human-mediated habitat change.
doi:10.1038/nature10574
PMCID: PMC4070744  PMID: 22048313
20.  Lipopolysaccharide differentially decreases plasma acyl and desacyl ghrelin levels in rats: potential role of the circulating ghrelin acylating enzyme GOAT 
Peptides  2010;31(9):1689-1696.
Bacterial lipopolysaccharide (LPS) in rodents is an established model for studying innate immune responses to gram-negative bacteria and mimicking symptoms of infections including reduced food intake associated with decreased circulating total ghrelin levels. The ghrelin-acylating enzyme, ghrelin-O-acyltransferase (GOAT) involved in the formation of acyl ghrelin (AG) was recently identified. We investigated changes in circulating AG, desacyl ghrelin (DG) and GOAT induced by intraperitoneal LPS (100μg/kg) and associated changes in food intake. Plasma AG and total ghrelin were assessed by radioimmunoassay, GOAT protein by Western blot and mRNA by RT-qPCR. DG was derived from total minus AG. Plasma AG and DG were decreased at 2h, 5h and 7h (p<0.01) post injection compared to vehicle and recovered at 24h. At 2h there was a significantly greater decrease of AG (-53%) than DG (-28%) resulting in a decreased AG/DG ratio (1:5, p <0.01), which thereafter returned to pre-injection values (1:3). This altered ratio was associated with a 38% decrease in plasma GOAT protein compared to vehicle (p <0.001), whereas gastric GOAT protein was slightly increased by 10% (p<0.05). GOAT mRNA expression was unchanged. Food intake was reduced by 58% measured during the 1.5-2h period post LPS injection. Decreased plasma AG and DG preceded the rise in rectal temperature and blood glucose that peaked at 7h. These data indicate that LPS induces a long-lasting reduction of AG and DG levels that may have a bearing with the decrease in food intake. The faster drop in AG than DG within 2h is associated with reduced circulating GOAT.
doi:10.1016/j.peptides.2010.06.015
PMCID: PMC4067316  PMID: 20599577
acyl ghrelin; desacyl ghrelin; GOAT; LPS; RAPID method; rat
21.  Lipopolysaccharide increases gastric and circulating NUCB2/nesfatin-1 concentrations in rats 
Peptides  2011;32(9):1942-1947.
Bacterial lipopolysaccharide (LPS) is an established animal model to study the innate immune response to Gram-negative bacteria mimicking symptoms of infection including reduction of food intake. LPS decreases acyl ghrelin associated with decreased concentrations of circulating ghrelin-O-acyltransferase (GOAT) likely contributing to the anorexigenic effect. We also recently described the prominent expression of the novel anorexigenic hormone, nucleobindin2 (NUCB2)/nesfatin-1 in gastric X/A-like cells co-localized with ghrelin in different pools of vesicles. To investigate whether LPS would affect gastric and circulating NUCB2/nesfatin-1 concentration, ad libitum fed rats were equipped with an intravenous (iv) catheter. LPS was injected intraperitoneally (ip, 100 μg/kg) and blood was withdrawn before and at 2, 5, 7 and 24 h post injection and processed for NUCB2/nesfatin-1 radioimmunoassay. Gastric corpus was collected to measure NUCB2 mRNA expression by RT-qPCR and NUCB2/nesfatin-1 protein concentration by Western blot. Injection of LPS increased plasma NUCB2/nesfatin-1 concentrations by 43%, 78% and 62% compared to vehicle at 2 h, 5 h and 7 h post injection respectively (p < 0.05) and returned to baseline at 24 h. The plasma NUCB2/nesfatin-1 increase at 2 h was associated with increased corpus NUCB2 mRNA expression (p < 0.01), whereas NUCB2 mRNA was not detectable in white blood cells. Likewise, gastric NUCB2 protein concentration was increased by 62% after LPS compared to vehicle (p < 0.01). These data show that gastric NUCB2 production and release are increased in response to LPS. These changes are opposite to those of ghrelin in response to LPS supporting a differential gastric regulation of NUCB2/nesfatin-1 and ghrelin expression derived from the same cell by immune challenge.
doi:10.1016/j.peptides.2011.07.006
PMCID: PMC4057044  PMID: 21782869
Endotoxin; Ghrelin; Nucleobindin2; Stomach; X/A-like cell
22.  Novel insight in distribution of nesfatin-1 and phospho-mTOR in the arcuate nucleus of the hypothalamus of rats 
Peptides  2009;31(2):257-262.
Recently, two proteins have been localized in the arcuate nucleus (ARC) and implicated in the regulation of food intake: the serine-threonine-kinase mammalian target of rapamycin (mTOR) as part of the TOR signaling complex 1 (TORC1), and nesfatin-1 derived from the precursor protein nucleobindin2. However, the exact cell types are not well described. Therefore, we performed double-labeling studies for NPY, CART, nesfatin-1 and pmTOR in the ARC. In this study, we showed that nesfatin-1 is not only intracellularly co-localized with cocaine- and amphetamine-regulated transcript (CART) peptide as reported before, but also with phospho-mTOR (pmTOR) and neuropeptide Y (NPY) in ARC neurons. Quantification revealed that 59 ± 5% of the pmTOR-immunoreactive (ir) neurons were immunoreactive for nesfatin-1. Moreover, double labeling for nesfatin-1 and NPY exhibited that 19 ± 5% of the NPY positive cells were also immunoreactive for nesfatin-1. Furthermore, we could also confirm results from previous studies, showing that the majority of nesfatin-1 neurons are also positive for CART peptide, whereas most of the pmTOR is co-localized with NPY and only to a lesser extent with CART.
doi:10.1016/j.peptides.2009.11.024
PMCID: PMC4043136  PMID: 19961888
Arcuate nucleus; Rat; Nesfatin-1; Phospho-mTOR; CART; NPY
23.  Vitamin C Deficiency in an Anticoagulated Patient 
ABSTRACT
A 64-year-old woman presented with a hemorrhagic perifollicular rash on her legs while taking warfarin. After biopsy, vitamin C deficiency was suggested as the diagnosis, which ascorbic acid assays later confirmed. Clinical resolution of the rash followed supplementation with vitamin C. Patients on a vitamin K limited diet may also be limiting their intake of vitamin C. Physicians should be aware of this possible correlation, and consider checking vitamin C levels in patients with a perifollicular hemorrhagic rash or other signs of vitamin C deficiency while on warfarin.
doi:10.1007/s11606-012-2282-3
PMCID: PMC3663949  PMID: 23192448
scurvy; vitamin C deficiency; warfarin; vitamin K
24.  Peripherally injected CCK-8S activates CART positive neurons of the paraventricular nucleus in rats 
Peptides  2010;31(6):1118-1123.
Cholecystokinin (CCK) plays a role in the short-term inhibition of food intake. Cocaine- and amphetamine-regulated transcript (CART) peptide has been observed in neurons of the paraventricular nucleus (PVN). It has been reported that intracerebroventricular injection of CART peptide inhibits food intake in rodents. The aim of the study was to determine whether intraperitoneally (ip) injected CCK-8S affects neuronal activity of PVN-CART neurons. Ad libitum fed male Sprague-Dawley rats received 6 or 10 μg/kg CCK-8S or 0.15 M NaCl ip (n = 4/group). The number of c-Fos-immunoreactive neurons was determined in the PVN, arcuate nucleus (ARC), and the nucleus of the solitary tract (NTS). CCK-8S dose-dependently increased the number of c-Fos-immunoreactive neurons in the PVN (mean ± SEM: 102 ± 6 vs. 150 ± 5 neurons/section, p < 0.05) and compared to vehicle treated rats (18 ± 7, p < 0.05 vs. 6 and 10 μg/kg CCK-8S). CCK-8S at both doses induced an increase in the number of c-Fos-immunoreactive neurons in the NTS (65 ± 13, p < 0.05, and 182 ± 16, p < 0.05). No effect on the number of c-Fos neurons was observed in the ARC. Immunostaining for CART and c-Fos revealed a dose-dependent increase of activated CART neurons (19 ± 3 vs. 29 ± 7; p < 0.05), only few activated CART neuron were observed in the vehicle group (1 ± 0). The present observation shows that CCK-8S injected ip induces an increase in neuronal activity in PVN-CART neurons and suggests that CART neurons in the PVN may play a role in the mediation of peripheral CCK-8S's anorexigenic effects.
doi:10.1016/j.peptides.2010.03.013
PMCID: PMC4040251  PMID: 20307613
CCK; CART; c-Fos; Food intake; PVN
25.  Sulfated cholecystokinin-8 activates phospho-mTOR immunoreactive neurons of the paraventricular nucleus in rats 
Peptides  2010;32(1):65-70.
The serin/threonin-kinase, mammalian target of rapamycin (mTOR) was detected in the arcuate nucleus (ARC) and paraventricular nucleus of the hypothalamus (PVN) and suggested to play a role in the integration of satiety signals. Since cholecystokinin (CCK) plays a role in the short-term inhibition of food intake and induces c-Fos in PVN neurons, the aim was to determine whether intraperitoneally injected CCK-8S affects the neuronal activity in cells immunoreactive for phospho-mTOR in the PVN. Ad libitum fed male Sprague-Dawley rats received 6 or 10 μg/kg CCK-8S or 0.15 M NaCl ip (n=4/group). The number of c-Fosimmunoreactive (ir) neurons was assessed in the PVN, ARC and in the nucleus of the solitary tract (NTS). CCK-8S increased the number of c-Fos-ir neurons in the PVN (6 μg: 103 ± 13 vs. 10 μg: 165 ± 14 neurons/section; p<0.05) compared to vehicle treated rats (4 ± 1, p<0.05), but not in the ARC. CCK-8S also dose-dependently increased the number of c-Fos neurons in the NTS. Staining for phospho-mTOR and c-Fos in the PVN showed a dose-dependent increase of activated phospho-mTOR neurons (17 ± 3 vs. 38 ± 2 neurons/section; p<0.05), while no activated phospho-mTOR neurons were observed in the vehicle group. Triple staining in the PVN showed activation of phospho-mTOR neurons co-localized with oxytocin, corresponding to 9.8 ± 3.6% and 19.5 ± 3.3% of oxytocin neurons respectively. Our observations indicate that peripheral CCK-8S activates phospho-mTOR neurons in the PVN and suggest that phospho-mTOR plays a role in the mediation of CCK-8S's anorexigenic effects.
doi:10.1016/j.peptides.2010.09.025
PMCID: PMC4040259  PMID: 20933028
CCK; c-Fos oxytocin; phospho-mTOR; PVN

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