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2.  Extensive Error in the Number of Genes Inferred from Draft Genome Assemblies 
PLoS Computational Biology  2014;10(12):e1003998.
Current sequencing methods produce large amounts of data, but genome assemblies based on these data are often woefully incomplete. These incomplete and error-filled assemblies result in many annotation errors, especially in the number of genes present in a genome. In this paper we investigate the magnitude of the problem, both in terms of total gene number and the number of copies of genes in specific families. To do this, we compare multiple draft assemblies against higher-quality versions of the same genomes, using several new assemblies of the chicken genome based on both traditional and next-generation sequencing technologies, as well as published draft assemblies of chimpanzee. We find that upwards of 40% of all gene families are inferred to have the wrong number of genes in draft assemblies, and that these incorrect assemblies both add and subtract genes. Using simulated genome assemblies of Drosophila melanogaster, we find that the major cause of increased gene numbers in draft genomes is the fragmentation of genes onto multiple individual contigs. Finally, we demonstrate the usefulness of RNA-Seq in improving the gene annotation of draft assemblies, largely by connecting genes that have been fragmented in the assembly process.
Author Summary
The initial publication of the genome sequence of many plants, animals, and microbes is often accompanied with great fanfare. However, these genomes are almost always first-drafts, with a lot of missing data, many gaps, and many errors in the published sequences. Compounding this problem, the genes identified in draft genome sequences are also affected by incomplete genome assemblies: the number and exact structure of predicted genes may be incorrect. Here we quantify the extent of such errors, by comparing several draft genomes against completed versions of the same sequences. Surprisingly, we find huge numbers of errors in the number of genes predicted from draft assemblies, with more than half of all genes having the wrong number of copies in the draft genomes examined. Our investigation also reveals the major causes of these errors, and further analyses using additional functional data demonstrate that many of the gene predictions can be corrected. The results presented here suggest that many inferences based on published draft genomes may be erroneous, but offer a way forward for future analyses.
doi:10.1371/journal.pcbi.1003998
PMCID: PMC4256071  PMID: 25474019
3.  Nerve growth factor acts through the TrkA receptor to protect sensory neurons from the damaging effects of the HIV-1 viral protein, Vpr 
Neuroscience  2013;252:10.1016/j.neuroscience.2013.07.046.
Distal sensory polyneuropathy (DSP) with associated neuropathic pain is the most common neurological disorder affecting patients with human immunodeficiency virus/acquired immunodeficiency syndrome (HIV/AIDS). Viral protein R (Vpr) is a neurotoxic protein encoded by HIV-1 and secreted by infected macrophages. Vpr reduces neuronal viability, increases cytosolic calcium and membrane excitability of cultured dorsal root ganglion (DRG) sensory neurons, and is associated with mechanical allodynia in vivo. A clinical trial with HIV/AIDS patients demonstrated that nerve growth factor (NGF) reduced the severity of DSP-associated neuropathic pain, a problem linked to damage to small diameter, potentially NGF responsive fibers. Herein, the actions of NGF were investigated in our Vpr model of DSP and we demonstrated that NGF significantly protected sensory neurons from the effects of Vpr. Footpads of immunodeficient Vpr transgenic (vpr/RAG1−/−) mice displayed allodynia (p<0.05), diminished epidermal innervation (p<0.01) and reduced NGF mRNA expression (p<0.001) compared to immunodeficient (wildtype/RAG1−/−) littermate control mice. Compartmented cultures confirmed recombinant Vpr exposure to the DRG neuronal perikarya decreased distal neurite extension (p<0.01), whereas NGF exposure at these distal axons protected the DRG neurons from the Vpr-induced effect on their cell bodies. NGF prevented Vpr-induced attenuation of the phosphorylated glycogen synthase-3 axon extension pathway and tropomyosin related kinase A (TrkA) receptor expression in DRG neurons (p<0.05) and it directly counteracted the cytosolic calcium burst caused by Vpr exposure to DRG neurons (p<0.01). TrkA receptor antagonists indicated that NGF acted through the TrkA receptor to block the Vpr-mediated decrease in axon outgrowth in neonatal and adult rat and fetal human DRG neurons (p<0.05). Similarly, inhibiting the lower affinity NGF receptor, p75, blocked Vpr’s effect on DRG neurons. Overall, NGF/TrkA signalling or p75 receptor inhibition protects somatic sensory neurons exposed to Vpr, thus laying the groundwork for potential therapeutic options for HIV/AIDS patients suffering from DSP.
doi:10.1016/j.neuroscience.2013.07.046
PMCID: PMC3829629  PMID: 23912036
4.  Capsule endoscopy capture rate: Has 4 frames-per-second any impact over 2 frames-per-second? 
World Journal of Gastroenterology : WJG  2014;20(39):14472-14478.
AIM: To compare the current capsule and a new prototype at 2 and 4 frames-per-second, respectively, in terms of clinical and therapeutic impact.
METHODS: One hundred patients with an indication for capsule endoscopy were included in the study. All procedures were performed with the new device (SB24). After an exhaustive evaluation of the SB24 videos, they were then converted to “SB2-like” videos for their evaluation. Findings, frames per finding, and clinical and therapeutic impact derived from video visualization were analyzed. Kappa index for interobserver agreement and χ2 and Student’s t tests for qualitative/quantitative variables, respectively, were used. Values of P under 0.05 were considered statistically significant.
RESULTS: Eighty-nine out of 100 cases included in the study were ultimately included in the analysis. The SB24 videos detected the anatomical landmarks (Z-line and duodenal papilla) and lesions in more patients than the “SB2-like” videos. On the other hand, the SB24 videos detected more frames per landmark/lesion than the “SB2-like” videos. However, these differences were not statistically significant (P > 0.05). Both clinical and therapeutic impacts were similar between SB24 and “SB2-like” videos (K = 0.954). The time spent by readers was significantly higher for SB24 videos visualization (P < 0.05) than for “SB2-like” videos when all images captured by the capsule were considered. However, these differences become non-significant if we only take into account small bowel images (P > 0.05).
CONCLUSION: More frames-per-second detect more landmarks, lesions, and frames per landmark/lesion, but is time consuming and has a very low impact on clinical and therapeutic management.
doi:10.3748/wjg.v20.i39.14472
PMCID: PMC4202376  PMID: 25339834
Capsule endoscopy; Small bowel; Diagnostic accuracy; Frames; Lesions; Landmarks
5.  Benefits of awake uniportal pulmonary resection in a patient with a previous contralateral lobectomy 
Surgical resection of a contralateral recurrence of non-small cell lung cancer (NSCLC) is indicated in patients without evidence of disseminated disease and considered functionally operable. General anesthesia and double-lumen intubation involves one lobe ventilation in a patient treated with a previous lobectomy, thus increasing the risks of ventilator-induced injuries and the morbidity. Awake procedures facilitate the surgery decreasing the anesthetic and surgical times, keeping the diaphragm motion and diminishing the ventilator-induced injuries into the remaining contralateral lobe. We present a 43-year-old woman with a previous left-lower lobectomy for a 3.1-cm mucinous adenocarcinoma 15 months before without nodal involvement, who presents a right-lower lobe 8-mm cavitated nodule, with evident radiological growth and fine-needle aspiration concordant with mucinous adenocarcinoma. We suggest an awake procedure with locoregional epidural anesthesia.
doi:10.3978/j.issn.2305-5839.2014.05.07
PMCID: PMC4205875  PMID: 25405168
Thoracoscopy/VATS; lung cancer surgery; anesthesia
6.  Cerebellar and parkinsonian phenotypes in multiple system atrophy: Similarities, differences and survival 
Multiple system atrophy (MSA) is a neurodegenerative disease with two motor phenotypes: parkinsonian (MSA-P) and cerebellar (MSA-C). To elucidate whether in addition to the motor abnormalities there are other significant differences between these phenotypes we performed a retrospective review of 100 patients (61 males, 39 females) with a diagnosis of possible (12%), or probable (88%). Four patients eventually had postmortem confirmation (i.e., definite MSA). Sixty percent were classified as having MSAP and 40% as MSA-C. MSA-C and MSA-P patients had similar male prevalence (60%), age of onset (56±9 years), and frequency of OH (69%). Brain MRI abnormalities were more frequent in MSA-C patients (p<0.001). Mean survival was 8±3 years for MSA-C and 9±4 years for MSA-P patients (p=0.22). Disease onset before 55 years predicted longer survival in both phenotypes. Initial autonomic involvement did not influence survival. We conclude that patients with both motor phenotypes have mostly similar survivals and demographic distributions. The differences here identified could help counseling of patients with MSA.
doi:10.1007/s00702-013-1133-7
PMCID: PMC4134009  PMID: 24337696
MSA; MSA-P; MSA-C; parkinsonism; cerebellar; autonomic; MRI
7.  Early-life stress, corpus callosum development, hippocampal volumetrics, and anxious behavior in male nonhuman primates 
Psychiatry research  2011;192(1):37-44.
Male bonnet monkeys (Macaca radiata) were subjected to the Variable Foraging Demand (VFD) early stress paradigm as infants, MRI scans were completed an average of four years later, and behavioral assessments of anxiety and ex-vivo corpus callosum (CC) measurements were made when animals were fully matured. VFD rearing was associated with smaller CC size, CC measurements were found to correlate with fearful behavior in adulthood, and ex-vivo CC assessments showed high consistency with earlier MRI measures. Region of Interest (ROI) hippocampus and whole brain voxel- based morphometry assessments were also completed and VFD rearing was associated with reduced hippocampus and inferior and middle temporal gyri volumes. Animals were also characterized according to serotonin transporter genotype (5-HTTLPR), and the effect of genotype on imaging parameters was explored. The current findings highlight the importance of future research to better understand the effects of stress on brain development in multiple regions, including the corpus callosum, hippocampus, and other regions involved in emotion processing. Nonhuman primates provide a powerful model to unravel the mechanisms by which early stress and genetic makeup interact to produce long-term changes in brain development, stress reactivity, and risk for psychiatric disorders.
doi:10.1016/j.pscychresns.2010.11.006
PMCID: PMC4090111  PMID: 21377844
stress; monkeys; corpus callosum; hippocampus; brain abnormalities; 5-HTTLPR
8.  The Impact of Extra-Domain Structures and Post-Translational Modifications in the Folding/Misfolding Behaviour of the Third PDZ Domain of MAGUK Neuronal Protein PSD-95 
PLoS ONE  2014;9(5):e98124.
The modulation of binding affinities and specificities by post-translational modifications located out from the binding pocket of the third PDZ domain of PSD-95 (PDZ3) has been reported recently. It is achieved through an intra-domain electrostatic network involving some charged residues in the β2–β3 loop (were a succinimide modification occurs), the α3 helix (an extra-structural element that links the PDZ3 domain with the following SH3 domain in PSD-95, and contains the phosphorylation target Tyr397), and the ligand peptide. Here, we have investigated the main structural and thermodynamic aspects that these structural elements and their related post-translational modifications display in the folding/misfolding pathway of PDZ3 by means of site-directed mutagenesis combined with calorimetry and spectroscopy. We have found that, although all the assayed mutations generate proteins more prone to aggregation than the wild-type PDZ3, those directly affecting the α3 helix, like the E401R substitution or the truncation of the whole α3 helix, increase the population of the DSC-detected intermediate state and the misfolding kinetics, by organizing the supramacromolecular structures at the expense of the two β-sheets present in the PDZ3 fold. However, those mutations affecting the β2–β3 loop, included into the prone-to-aggregation region composed by a single β-sheet comprising β2 to β4 chains, stabilize the trimeric intermediate previously shown in the wild-type PDZ3 and slow-down aggregation, also making it partly reversible. These results strongly suggest that the α3 helix protects to some extent the PDZ3 domain core from misfolding. This might well constitute the first example where an extra-element, intended to link the PDZ3 domain to the following SH3 in PSD-95 and in other members of the MAGUK family, not only regulates the binding abilities of this domain but it also protects PDZ3 from misfolding and aggregation. The influence of the post-translational modifications in this regulatory mechanism is also discussed.
doi:10.1371/journal.pone.0098124
PMCID: PMC4028313  PMID: 24845085
9.  Hyperdopaminergic crises in familial dysautonomia 
Neurology  2013;80(17):1611-1617.
Objective:
The purpose of this study was to determine whether carbidopa (Lodosyn), an inhibitor of dopa-decarboxylase that blocks the synthesis of dopamine outside the brain, is an effective antiemetic in patients with familial dysautonomia (FD) and hyperdopaminergic nausea/retching/vomiting attacks.
Methods:
We enrolled 12 patients with FD in an open-label titration and treatment study to assess the safety of carbidopa. We then conducted a randomized, double-blind, placebo-controlled, crossover study to evaluate its antiemetic efficacy.
Results:
Previous fundoplication surgery in each patient studied prevented vomiting, but all of the subjects experienced severe cyclical nausea and uncontrollable retching that was refractory to standard treatments. Carbidopa at an average daily dose of 480 mg (range 325–600 mg/day) was well tolerated. In the double-blind phase, patients experienced significantly less nausea and retching while on carbidopa than on placebo (p < 0.03 and p < 0.02, respectively). Twenty-four-hour urinary dopamine excretion was significantly lower while on carbidopa (147 ± 32 µg/gCr) than while on placebo (222 ± 41µg/gCr, p < 0.05).
Conclusions:
Carbidopa is a safe and effective antiemetic in patients with FD, likely by reducing the formation of dopamine outside the brain.
Classification of evidence:
This study provides Class II evidence that carbidopa is effective in reducing nausea/retching/vomiting in patients with FD.
doi:10.1212/WNL.0b013e31828f18f0
PMCID: PMC3662326  PMID: 23553478
11.  Post-Translational Modifications Modulate Ligand Recognition by the Third PDZ Domain of the MAGUK Protein PSD-95 
PLoS ONE  2014;9(2):e90030.
The relative promiscuity of hub proteins such as postsynaptic density protein-95 (PSD-95) can be achieved by alternative splicing, allosteric regulation, and post-translational modifications, the latter of which is the most efficient method of accelerating cellular responses to environmental changes in vivo. Here, a mutational approach was used to determine the impact of phosphorylation and succinimidation post-translational modifications on the binding affinity of the postsynaptic density protein-95/discs large/zonula occludens-1 (PDZ3) domain of PSD-95. Molecular dynamics simulations revealed that the binding affinity of this domain is influenced by an interplay between salt-bridges linking the α3 helix, the β2–β3 loop and the positively charged Lys residues in its high-affinity hexapeptide ligand KKETAV. The α3 helix is an extra structural element that is not present in other PDZ domains, which links PDZ3 with the following SH3 domain in the PSD-95 protein. This regulatory mechanism was confirmed experimentally via thermodynamic and NMR chemical shift perturbation analyses, discarding intra-domain long-range effects. Taken together, the results presented here reveal the molecular basis of the regulatory role of the α3 extra-element and the effects of post-translational modifications of PDZ3 on its binding affinity, both energetically and dynamically.
doi:10.1371/journal.pone.0090030
PMCID: PMC3935999  PMID: 24587199
12.  Evaluation of Epidemiological Cut-Off Values Indicates that Biocide Resistant Subpopulations Are Uncommon in Natural Isolates of Clinically-Relevant Microorganisms 
PLoS ONE  2014;9(1):e86669.
To date there are no clear criteria to determine whether a microbe is susceptible to biocides or not. As a starting point for distinguishing between wild-type and resistant organisms, we set out to determine the minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) distributions for four common biocides; triclosan, benzalkonium chloride, chlorhexidine and sodium hypochlorite for 3319 clinical isolates, with a particular focus on Staphylococcus aureus (N = 1635) and Salmonella spp. (N = 901) but also including Escherichia coli (N = 368), Candida albicans (N = 200), Klebsiella pneumoniae (N = 60), Enterobacter spp. (N = 54), Enterococcus faecium (N = 53), and Enterococcus faecalis (N = 56). From these data epidemiological cut-off values (ECOFFs) are proposed. As would be expected, MBCs were higher than MICs for all biocides. In most cases both values followed a normal distribution. Bimodal distributions, indicating the existence of biocide resistant subpopulations were observed for Enterobacter chlorhexidine susceptibility (both MICs and MBCs) and the susceptibility to triclosan of Enterobacter (MBC), E. coli (MBC and MIC) and S. aureus (MBC and MIC). There is a concern on the potential selection of antibiotic resistance by biocides. Our results indicate however that resistance to biocides and, hence any potential association with antibiotic resistance, is uncommon in natural populations of clinically relevant microorganisms.
doi:10.1371/journal.pone.0086669
PMCID: PMC3900580  PMID: 24466194
13.  Transcriptional and biochemical responses of monoacylglycerol acyltransferase-mediated oil synthesis and associated senescence-like responses in Nicotiana benthamiana 
Triacylglycerol (TAG) accumulates in plant seeds as a major renewable source of carbon for food, fuel and industrial feedstock. Approaches to enhance TAG content by altering lipid pathways and genes in vegetative parts have gained significant attention for biofuel and other applications. However, consequences of these modifications are not always studied in detail. In an attempt to increase TAG levels in leaves we previously demonstrated that a novel substrate, monoacylglycerol (MAG), can be used for the biosynthesis of diacylglycerol (DAG) and TAG. Transient expression of the Mus musculus monoacylglycerol acyltransferases MGAT1 and 2 in the model plant Nicotiana benthamiana increased TAG levels at 5 days post-infiltration (dpi). Here we show that increased TAG and DAG levels can be achieved as early as 2 dpi. In addition, the MGAT1 infiltrated areas showed senescence-like symptoms from 3 dpi onwards. To unravel underlying molecular mechanisms, Illumina deep sequencing was carried out (a) for de-novo assembling and annotation of N. benthamiana leaf transcripts and (b) to characterize MGAT1-responsive transcriptome. We found that MGAT1-responsive genes are involved in several processes including TAG biosynthesis, photosynthesis, cell-wall, cutin, suberin, wax and mucilage biosynthesis, lipid and hormone metabolism. Comparative analysis with transcript profiles from other senescence studies identified characteristic gene expression changes involved in senescence induction. We confirmed that increased TAG and observed senescence-symptoms are due to the MAG depletion caused by MGAT1 activity and suggest a mechanism for MGAT1 induced TAG increase and senescence-like symptoms. The data generated will serve as a valuable resource for oil and senescence related studies and for future N. benthamiana transcriptome studies.
doi:10.3389/fpls.2014.00204
PMCID: PMC4033622  PMID: 24904604
Nicotiana benthamiana; monoacylglycerol; diacylglycerol; triacylglycerol; acyltransferase; oil increase; senescence; differential gene expression
14.  Early life stress and macaque amygdala hypertrophy: preliminary evidence for a role for the serotonin transporter gene 
Background: Children exposed to early life stress (ELS) exhibit enlarged amygdala volume in comparison to controls. The primary goal of this study was to examine amygdala volumes in bonnet macaques subjected to maternal variable foraging demand (VFD) rearing, a well-established model of ELS. Preliminary analyses examined the interaction of ELS and the serotonin transporter gene on amygdala volume. Secondary analyses were conducted to examine the association between amygdala volume and other stress-related variables previously found to distinguish VFD and non-VFD reared animals.
Methods: Twelve VFD-reared and nine normally reared monkeys completed MRI scans on a 3T system (mean age = 5.2 years).
Results: Left amygdala volume was larger in VFD vs. control macaques. Larger amygdala volume was associated with: “high” cerebrospinal fluid concentrations of corticotropin releasing-factor (CRF) determined when the animals were in adolescence (mean age = 2.7 years); reduced fractional anisotropy (FA) of the anterior limb of the internal capsule (ALIC) during young adulthood (mean age = 5.2 years) and timid anxiety-like responses to an intruder during full adulthood (mean age = 8.4 years). Right amygdala volume varied inversely with left hippocampal neurogenesis assessed in late adulthood (mean age = 8.7 years). Exploratory analyses also showed a gene-by-environment effect, with VFD-reared macaques with a single short allele of the serotonin transporter gene exhibiting larger amygdala volume compared to VFD-reared subjects with only the long allele and normally reared controls.
Conclusion: These data suggest that the left amygdala exhibits hypertrophy after ELS, particularly in association with the serotonin transporter gene, and that amygdala volume variation occurs in concert with other key stress-related behavioral and neurobiological parameters observed across the lifecycle. Future research is required to understand the mechanisms underlying these diverse and persistent changes associated with ELS and amygdala volume.
doi:10.3389/fnbeh.2014.00342
PMCID: PMC4186477  PMID: 25339875
amygdala; early life stress; non-human primates; MRI; stress; serotonin transporter gene
15.  SERPINE1, PAI-1 protein coding gene, methylation levels and epigenetic relationships with adiposity changes in obese subjects with metabolic syndrome features under dietary restriction 
Plasminogen activator inhibitor 1 (PAI-1) has been associated with metabolic disorders, through different mechanisms, which could involve changes in DNA methylation. This work aimed to assess the potential relationships of the cytosine methylation levels within SERPINE1 gene transcriptional regulatory region, which codes for PAI-1, in peripheral white blood cells with anthropometrical, metabolic and inflammatory features. Forty-six obese subjects with metabolic syndrome features followed Control or Metabolic Syndrome Reduction in Navarra (RESMENA) energy-restricted (−30%E) diets for 8 weeks. SERPINE1 transcriptional regulatory region methylation at baseline was analyzed by a microarray technical. Both dietary strategies reduced anthropometric and biochemical parameters. The Control group significantly reduced plasma PAI-1 concentrations but not the RESMENA group. Participants from both nutritional interventions with higher SERPINE1 methylation levels at baseline showed significantly major reductions in body weight, total fat mass, android fat mass, total cholesterol and triglycerides, as compared with those with lower initial SERPINE1 methylation levels. In conclusion, the DNA methylation levels of SERPINE1 transcriptional regulatory region were associated with some metabolic and anthropometric changes in obese subjects with metabolic syndrome under energy restriction, suggesting a complex epigenetic network in the regulation of this recognized pro-inflammatory marker. (www.clinicaltrials.gov; NCT01087086)
doi:10.3164/jcbn.13-54
PMCID: PMC3818272  PMID: 24249967
DNA methylation; PAI-1; SERPINE1 gene; metabolic syndrome; energy restriction
16.  Loss of Innervation and Axon Plasticity Accompanies Impaired Diabetic Wound Healing 
PLoS ONE  2013;8(9):e75877.
Loss of cutaneous innervation from sensory neuropathy is included among mechanisms for impaired healing of diabetic skin wounds. The relationships between cutaneous axons and their local microenvironment during wound healing are challenged in diabetes. Here, we show that secondary wound closure of the hairy dorsal skin of mice is delayed by diabetes and is associated with not only a pre-existing loss of cutaneous axons but substantial retraction of axons around the wound. At 7d following a 3mm punch wound, a critical period of healing and reinnervation, both intact skin nearby the wound and skin directly at the wound margins had over 30-50% fewer axons and a larger deficit of ingrowing axons in diabetics. These findings contrasted with a pre-existing 10-15% deficit in axons. Moreover, new diabetic ingrowing axons had less evidence of plasticity. Unexpectedly, hair follicles adjacent to the wounds had a 70% reduction in their innervation associated with depleted expression of hair follicular stem cell markers. These impairments were associated with the local upregulation of two established axon regenerative ‘roadblocks’: PTEN and RHOA, potential but thus far unexplored mediators of these changes. The overall findings identify striking and unexpected superimposed cutaneous axon loss or retraction beyond that expected of diabetic neuropathy alone, associated with experimental diabetic skin wounding, a finding that prompts new considerations in diabetic wounds.
doi:10.1371/journal.pone.0075877
PMCID: PMC3786937  PMID: 24098736
17.  Peritoneal Dissemination of Hepatoid Carcinoma of the Ovary Treated with Cytoreductive Surgery and Hyperthermic Intraoperative Intraperitoneal Chemotherapy 
Case Reports in Medicine  2013;2013:283295.
Hepatoid carcinoma of the ovary (HCO) was first reported in 1987 in 5 cases of malignant ovarian tumors which were similar to hepatocarcinoma in the histological analysis. We report the first case in the literature of a patient diagnosed with HCO treated using HIPEC after extensive cytoreductive surgery, and we discuss the value of this therapeutical option in patients with HCO.
doi:10.1155/2013/283295
PMCID: PMC3691891  PMID: 23840218
19.  A clinical exploratory study with itolizumab, an anti-CD6 monoclonal antibody, in patients with rheumatoid arthritis 
Results in Immunology  2012;2:204-211.
T cells are involved in the pathogenesis of rheumatoid arthritis (RA). CD6 is a co-stimulatory molecule, predominantly expressed on lymphocytes, that has been linked to autoreactive responses. The purpose of this study was to evaluate the safety, immunogenicity and preliminary efficacy of itolizumab, a humanized anti-CD6 monoclonal antibody, in patients with active rheumatoid arthritis. Fifteen patients were enrolled in a phase I, open-label, dose-finding study. Five cohorts of patients received a weekly antibody monotherapy with a dose-range from 0.1 to 0.8 mg/kg. Itolizumab showed a good safety profile, with no severe or serious adverse events reported so far. No signs or symptoms associated with immunosuppression were observed in the study. Objective clinical responses were achieved in more than 80% of patients after treatment completion, and these responses tend to be sustained afterwards. This clinical study constitutes the first evidence of the safety and positive clinical effect of a monotherapy using an anti-CD6 antibody in patients with rheumatoid arthritis.
Highlights
▸ Exploratory study to assess itolizumab in rheumatoid arthritis patients. ▸ Good safety profile, neither severe or serious adverse events, nor immunosuppression. ▸ Objective clinical response achieved in more than 80% of patients. ▸ Clinical response trends to be sustained after itolizumab withdrawal.
doi:10.1016/j.rinim.2012.11.001
PMCID: PMC3862386  PMID: 24371585
Rheumatoid arthritis; Exploratory study; T lymphocyte; CD6; ACR, American College of Rheumatology; AE, adverse events; CRP, C reactive protein; iv, intravenous; DMARD, disease-modifying antirheumatic drug; ESR, eritrosedimentation rate; mAbs, monoclonal antibodies; NSAIDs, nonsteroidal antiinflammatory drugs; RA, rheumatoid arthritis; RF, rheumatoid factor; SAE, serious adverse event.
20.  Cooperativity in RNA-Protein Interactions: Global Analysis of RNA Binding Specificity 
Cell reports  2012;1(5):570-581.
Summary
The control and function of RNA are governed by the specificity of RNA binding proteins. Here, we describe a method for global unbiased analysis of RNA-protein interactions that uses in vitro selection, high-throughput sequencing, and sequence-specificity landscapes. The method yields affinities for a vast array of RNAs in a single experiment, including both low- and high-affinity sites. It is reproducible and accurate. Using this approach, we analyzed members of the PUF (Pumilio and FBF) family of eukaryotic mRNA regulators. Our data identify effects of a specific protein partner on PUF-RNA interactions, reveal subsets of target sites not previously detected, and demonstrate that designer PUF proteins can precisely alter specificity. The approach described here is, in principle, broadly applicable for analysis of any molecule that binds RNA, including proteins, nucleic acids, and small molecules.
doi:10.1016/j.celrep.2012.04.003
PMCID: PMC3375920  PMID: 22708079
21.  Interspecific Hybridization Increased in Congeneric Flatfishes after the Prestige Oil Spill 
PLoS ONE  2012;7(4):e34485.
Marine species with relatively low migratory capacity are threatened by habitat alterations derived from human activities. In November 2002 the tanker Prestige sank off the Spanish northwest coast releasing 70,000 tons of fuel and damaging biota in the area. Despite efforts to clean the damaged areas, fuel remnants have affected marine species over the last nine years. This study is focused on two flatfish, Lepidorhombus boscii (four-spotted megrim) and L. whiffiagonis (megrim), whose spawning areas are located at the edge of the continental platform. We have analyzed megrim samples from North Spanish and French waters obtained before and after the oil spill. Genotypes at the nuclear marker 5S rDNA indicate a significant increase in interspecific hybridization after the Prestige accident, likely due to forced spawning overlap. The mitochondrial D-Loop region was employed for determining the direction of hybrid crosses, which were most frequently L. boscii female x L. whiffiagonis male. Reduced ability of L. boscii females to select conspecific mates would explain such asymmetric hybridization. To our knowledge this is the first time that increased hybridization between fish species can be associated to an oil spill. These results illustrate the potential long-term effect of petrol wastes on wild fish species.
doi:10.1371/journal.pone.0034485
PMCID: PMC3325272  PMID: 22511945
22.  284 Persistence of Nasal and Bronchial Symptoms in Patients with Samter's Syndrome with Treatment Medical and Surgical in a 2 Year Period 
The World Allergy Organization Journal  2012;5(Suppl 2):S109-S110.
Background
Know the causes of nasal and bronchial symptoms persistence in patients with Samter's syndrome under treatment in a period of time.
Methods
Cohort study. Inclusion criteria: Patients with asthma diagnoses, hypersensitivity to aspirin and nasal polyps. Exclusion criteria: Other kind of asthma, COPD. Twelve patients were followed from June 2009 to June 2011. Nasal and bronchial symptoms were assessed every 6 months using the Visual Analogue Scale of severity (VAS) from EPOS guidelines and spirometry from GINA. All were treated with intranasal mometasone furoate 200 mcg at day, montelukast 10 mg at day, salmeterol plus fluticasone 50/100 powder 2 inhalation every 12 hours, fluticasone spray 150 mg every 12 hours, loratadine tablet 10 mg if was necessary, with modifications of doses every 3 months.
Patients diagnosed at 6 months with sinusitis and nasal polyposis were administered amoxicillin plus clavulanate 1.5 g daily for 5 weeks. The patients without response at 6 and 18 months were prescribed clarithromycin 400 mg daily for 4 weeks. All patients underwent CT of the sinuses through the Lund-Mackay system, chest CT scan, skin prick test. Evaluated by otolaryngology at the 6, 12, and 18 months.
Results
In the 98, 2% had negative skin prick tests. At 6 months, 58.3% had nasal symptoms with VAS <7. At 33.3% reported bronchial relapses with FEV1 <80. At year nasal symptoms increased, with WAS> 7 in 66.6%. The bronchial relapse decreased to 16.6%. At year and a half it increased nasal symptoms in 75% of patients, with VAS> 7. At 41.6% had obstruction of 100% and pansinusitis. They needed antibiotic scheme. At 2 years in 83.3% had a VAS>7. At 58.3% had pansinusitis. The bronchial relapse did not increase. We determined the presence of VAS> 7 and pansinusitis (OR = 4). The bronchial relapse did not influence with increasing VAS (OR = 1).
Conclusions
Nasal symptoms persistent were secondary to the nasal polyps and pansinusitis with higher levels of VAS. It was determinated a 4-fold risk over pansinusitis with a VAS> 7 (OR = 4). It should be stressed the palliative surgical treatment in earlier stages and desensitization protocols.
doi:10.1097/01.WOX.0000412041.67199.e9
PMCID: PMC3512967
23.  Comparison of central versus peripheral delivery of pregabalin in neuropathic pain states 
Molecular Pain  2012;8:3.
Background
Although pregabalin therapy is beneficial for neuropathic pain (NeP) by targeting the CaVα2δ-1 subunit, its site of action is uncertain. Direct targeting of the central nervous system may be beneficial for the avoidance of systemic side effects.
Results
We used intranasal, intrathecal, and near-nerve chamber forms of delivery of varying concentrations of pregabalin or saline delivered over 14 days in rat models of experimental diabetic peripheral neuropathy and spinal nerve ligation. As well, radiolabelled pregabalin was administered to determine localization with different deliveries. We evaluated tactile allodynia and thermal hyperalgesia at multiple time points, and then analyzed harvested nervous system tissues for molecular and immunohistochemical changes in CaVα2δ-1 protein expression. Both intrathecal and intranasal pregabalin administration at high concentrations relieved NeP behaviors, while near-nerve pregabalin delivery had no effect. NeP was associated with upregulation of CACNA2D1 mRNA and CaVα2δ-1 protein within peripheral nerve, dorsal root ganglia (DRG), and dorsal spinal cord, but not brain. Pregabalin's effect was limited to suppression of CaVα2δ-1 protein (but not CACNA2D1 mRNA) expression at the spinal dorsal horn in neuropathic pain states. Dorsal root ligation prevented CaVα2δ-1 protein trafficking anterograde from the dorsal root ganglia to the dorsal horn after neuropathic pain initiation.
Conclusions
Either intranasal or intrathecal pregabalin relieves neuropathic pain behaviours, perhaps due to pregabalin's effect upon anterograde CaVα2δ-1 protein trafficking from the DRG to the dorsal horn. Intranasal delivery of agents such as pregabalin may be an attractive alternative to systemic therapy for management of neuropathic pain states.
doi:10.1186/1744-8069-8-3
PMCID: PMC3285045  PMID: 22236461
neuropathic pain; pregabalin; diabetic peripheral neuropathy; spinal nerve ligation
24.  Induction of the Aryl Hydrocarbon Receptor–Responsive Genes and Modulation of the Immunoglobulin M Response by 2,3,7,8-Tetrachlorodibenzo-p-Dioxin in Primary Human B Cells 
Toxicological Sciences  2010;118(1):86-97.
Past studies in rodent models identified the suppression of primary humoral immune responses as one of the most sensitive sequela associated with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) exposure. Yet, the sensitivity of humoral immunity to TCDD in humans represents an important toxicological data gap. Therefore, the objectives of this investigation were two-fold. The first was to assess the induction of known aryl hydrocarbon receptor (AHR)–responsive genes in primary human B cells as a measure of early biological responses to TCDD. The second was to evaluate the direct effect of TCDD on CD40 ligand–induced immunoglobulin M (IgM) secretion by human primary B cells. The effects of TCDD on induction of AHR-responsive genes and suppression of the IgM response were also compared with B cells from a TCDD-responsive mouse strain, C57BL/6. AHR-responsive genes in human B cells exhibited slower kinetics and reduced magnitude of induction by TCDD when compared with mouse B cells. Evaluation of B-cell function from 12 donors identified two general phenotypes; the majority of donors exhibited similar sensitivity to suppression by TCDD of the IgM response as mouse B cells, which was not attributable to decreased B-cell proliferation. In a minority of donors, no suppression of the IgM response by TCDD was observed. Although donor-to-donor variation in sensitivity to TCDD was observed, human B cells from the majority of donors evaluated showed impairment of effector function by TCDD. Collectively, data presented in this series of studies demonstrate that TCDD impairs the humoral immunity of humans by directly targeting B cells.
doi:10.1093/toxsci/kfq234
PMCID: PMC2955211  PMID: 20702590
TCDD; immunotoxicology; antibody response; human B cell; AHR
25.  The Coexistence of an Intrasellar Adenoma, Lymphocytic Hypophysitis, and Primary Pituitary Lymphoma in a Patient with Acromegaly 
Case Reports in Endocrinology  2011;2011:941738.
The concomitant presence of three histopathologically different entities in the pituitary gland is a rare occurrence. Most publications identify at least two distinct pathologies, mainly, a pituitary adenoma coexisting with a second intrasellar lesion. We present a case of a 71-year-old female referred for evaluation and treatment of acromegaly. Questioning revealed she was experiencing facial palsy, visual disturbances, and syncopal spells for several weeks. When laboratory evaluation showed elevated somatomedin (IGF-I) levels and an oral glucose tolerance test failed to demonstrate any suppression of her growth hormone (GH) values, an MRI of the pituitary revealed a sellar mass. A presumptive diagnosis of pituitary adenoma was established. The patient underwent transsphenoidal resection of the sellar mass, which proved to be a large B-cell lymphoma (Stage I-E) associated with areas of adenoma and lymphocytic hypophysitis.
doi:10.1155/2011/941738
PMCID: PMC3420670  PMID: 22937293

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