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1.  The influence of normative and subjective oral health status on schoolchildren’s happiness 
BMC Oral Health  2015;15:15.
Traditional methods to measure oral health based on clinical standards are limited because they do not consider psychosocial and functional aspects of oral health. It has been recommended that these measures need to be supplemented by data obtained from patients regarding their individual perceptions on oral health-related quality of life (OHRQoL). Happiness is a multidimensional construct comprising both emotional and cognitive domains, and has been defined as “the degree to which an individual judges the overall quality of his or her life as a whole favorably”. It has been associated with several health outcomes, including oral health. The aim of this study was to assess the impact of oral health conditions, oral health-related quality of life (OHRQoL), and socioeconomic factors on the subjective happiness of Brazilian adolescents.
A cross-sectional study was conducted in 2012 on a representative sample of 12-year-old schoolchildren in Santa Maria-RS, Brazil. The data were collected through dental examinations and structured interviews. The participants underwent an evaluation aimed at detecting dental caries, traumatic dental injuries, malocclusion, and gingival bleeding. They also completed the Brazilian versions of the Child Perceptions Questionnaire-short form (CPQ11–14 – ISF: 16) and the Subjective Happiness Scale (SHS), which was our outcome variable. Socioeconomic conditions were evaluated through a questionnaire that was completed by the participants’ parents. Poisson regression analysis was used to determine the association between the explanatory variables and the outcome. Moreover, a correlation analysis was performed to determine the relationship between the SHS scores and the overall and domain scores of the CPQ11–14 –ISF: 16.
A total of 1,134 children were evaluated. Unadjusted analyses showed that happiness was associated with socioeconomic indicators, the use of dental services, clinical status, and scores on the OHRQoL measure. After adjustment, household overcrowding (RR: 0.96; 95% CI: 0.93-0.98), dental caries (RR: 0.98; 95% CI: 0.97-0.99), malocclusion (RR: 0.98; 95% CI: 0.96-0.99), and the severity associated with the CPQ11-14 (RR: 0.95; 95% CI: 0.93-0.97) still showed a significant association with lower levels of the mean SHS score.
Happiness is influenced by oral conditions, socioeconomic status, and OHRQoL.
PMCID: PMC4320443  PMID: 25616978
Happiness; Child; Oral health; Quality of life
2.  Array-Based Comparative Genomic Hybridization Analysis Reveals Chromosomal Copy Number Aberrations Associated with Clinical Outcome in Canine Diffuse Large B-Cell Lymphoma 
PLoS ONE  2014;9(11):e111817.
Canine Diffuse Large B-cell Lymphoma (cDLBCL) is an aggressive cancer with variable clinical response. Despite recent attempts by gene expression profiling to identify the dog as a potential animal model for human DLBCL, this tumor remains biologically heterogeneous with no prognostic biomarkers to predict prognosis. The aim of this work was to identify copy number aberrations (CNAs) by high-resolution array comparative genomic hybridization (aCGH) in 12 dogs with newly diagnosed DLBCL. In a subset of these dogs, the genetic profiles at the end of therapy and at relapse were also assessed. In primary DLBCLs, 90 different genomic imbalances were counted, consisting of 46 gains and 44 losses. Two gains in chr13 were significantly correlated with clinical stage. In addition, specific regions of gains and losses were significantly associated to duration of remission. In primary DLBCLs, individual variability was found, however 14 recurrent CNAs (>30%) were identified. Losses involving IGK, IGL and IGH were always found, and gains along the length of chr13 and chr31 were often observed (>41%). In these segments, MYC, LDHB, HSF1, KIT and PDGFRα are annotated. At the end of therapy, dogs in remission showed four new CNAs, whereas three new CNAs were observed in dogs at relapse compared with the previous profiles. One ex novo CNA, involving TCR, was present in dogs in remission after therapy, possibly induced by the autologous vaccine. Overall, aCGH identified small CNAs associated with outcome, which, along with future expression studies, may reveal target genes relevant to cDLBCL.
PMCID: PMC4221131  PMID: 25372838
3.  Genetic adaptation of the human circadian clock to day-length latitudinal variations and relevance for affective disorders 
Genome Biology  2014;15(10):499.
The temporal coordination of biological processes into daily cycles is a common feature of most living organisms. In humans, disruption of circadian rhythms is commonly observed in psychiatric diseases, including schizophrenia, bipolar disorder, depression and autism. Light therapy is the most effective treatment for seasonal affective disorder and circadian-related treatments sustain antidepressant response in bipolar disorder patients. Day/night cycles represent a major circadian synchronizing signal and vary widely with latitude.
We apply a geographically explicit model to show that out-of-Africa migration, which led humans to occupy a wide latitudinal area, affected the evolutionary history of circadian regulatory genes. The SNPs we identify using this model display consistent signals of natural selection using tests based on population genetic differentiation and haplotype homozygosity. Signals of natural selection driven by annual photoperiod variation are detected for schizophrenia, bipolar disorder, and restless leg syndrome risk variants, in line with the circadian component of these conditions.
Our results suggest that human populations adapted to life at different latitudes by tuning their circadian clock systems. This process also involves risk variants for neuropsychiatric conditions, suggesting possible genetic modulators for chronotherapies and candidates for interaction analysis with photoperiod-related environmental variables, such as season of birth, country of residence, shift-work or lifestyle habits.
Electronic supplementary material
The online version of this article (doi:10.1186/s13059-014-0499-7) contains supplementary material, which is available to authorized users.
PMCID: PMC4237747  PMID: 25358694
4.  Albuminoid Genes: Evolving at the Interface of Dispensability and Selection 
Genome Biology and Evolution  2014;6(11):2983-2997.
The albuminoid gene family comprises vitamin D-binding protein (GC), alpha-fetoprotein (AFP), afamin (AFM), and albumin (ALB). Albumin is the most abundant human serum protein, and, as the other family members, acts as a transporter of endogenous and exogenous substances including thyroxine, fatty acids, and drugs. Instead, the major cargo of GC is 25-hydroxyvitamin D. We performed an evolutionary study of albuminoid genes and we show that ALB evolved adaptively in mammals. Most positively selected sites are located within albumin-binding sites for fatty acids and thyroxine, as well as at the contact surface with neonatal Fc receptor. Positive selection was also detected for residues forming the prostaglandin-binding pocket. Adaptation to hibernation/torpor might explain the signatures of episodic positive selection we detected for few mammalian lineages. Application of a population genetics–phylogenetics approach showed that purifying selection represented a major force acting on albuminoid genes in both humans and chimpanzees, with the strongest constraint observed for human GC. Population genetic analysis revealed that GC was also the target of locally exerted selective pressure, which drove the frequency increase of different haplotypes in distinct human populations. A search for known variants that modulate GC and 25-hydroxyvitamin D concentrations revealed linkage disequilibrium with positively selected variants, although European and Asian major GC haplotypes carry alleles with reported opposite effect on GC concentration. Data herein indicate that albumin, an extremely abundant housekeeping protein, was the target of pervasive and episodic selection in mammals, whereas GC represented a selection target during the recent evolution of human populations.
PMCID: PMC4255767  PMID: 25349266
albumin; vitamin D-binding protein; albuminoids; positive selection; vitamin D; hibernation
5.  Contribution of α,β-Amyrenone to the Anti-Inflammatory and Antihypersensitivity Effects of Aleurites moluccana (L.) Willd. 
BioMed Research International  2014;2014:636839.
The aim of the study was to analyze the constituents of the dichloromethane fraction obtained from A. moluccana and also to evaluate the anti-inflammatory and antinociceptive properties of α,β-amyrenone isolated from A. moluccana in mice. The dichloromethane fraction was evaluated by gas chromatography and submitted to purification. The mixture of α,β-amyrenone was isolated and then evaluated using the carrageenan-induced paw-oedema or pleurisy and CFA-induced arthritis models in mice. Five triterpenes, α,β-amyrenone, glutinol, and α,β-amyrin were isolated from dichloromethane fraction of A. moluccana leaf extract. The mixture of α,β-amyrenone, dosed orally, was able to reduce mechanical hypersensitivity and paw-oedema induced by carrageenan, interfering with neutrophil migration. Similar results were observed in the carrageenan-induced pleurisy model. Repeated administration of the compounds was also effective in reducing the mechanical sensitization and oedema developed in the arthritis model induced by CFA. In conclusion, the results demonstrate that α,β-amyrenone interferes in both acute and chronic inflammatory processes. We can infer that these effects involve, at least in part, a reduction in the neutrophil migration. Therefore, it seems reasonable to suggest that α,β-amyrenone could represent a new therapeutic tool for the management of painful and inflammatory diseases, especially those presenting a chronic profile.
PMCID: PMC4217248  PMID: 25386561
6.  Defective autophagy in spastizin mutated patients with hereditary spastic paraparesis type 15 
Brain  2013;136(10):3119-3139.
Hereditary spastic paraparesis type 15 is a recessive complicated form of the disease clinically characterized by slowly progressive spastic paraparesis and mental deterioration with onset between the first and second decade of life. Thinning of corpus callosum is the neuroradiological distinctive sign frequently associated with white matter abnormalities. The causative gene, ZFYVE26, encodes a large protein of 2539 amino acid residues, termed spastizin, containing three recognizable domains: a zinc finger, a leucine zipper and a FYVE domain. Spastizin protein has a diffuse cytoplasmic distribution and co-localizes partially with early endosomes, the endoplasmic reticulum, microtubules and vesicles involved in protein trafficking. In addition, spastizin localizes to the mid-body during the final step of mitosis and contributes to successful cytokinesis. Spastizin interacts with Beclin 1, a protein required for cytokinesis and autophagy, which is the major lysosome-mediated degradation process in the cell. In view of the Beclin 1–spastizin interaction, we investigated the possible role of spastizin in autophagy. We carried out this analysis by using lymphoblast and fibroblast cells derived from four different spastizin mutated patients (p.I508N, p.L243P, p.R1209fsX, p.S1312X) and from control subjects. Of note, the truncating p.R1209fsX and p.S1312X mutations lead to loss of spastizin protein. The results obtained indicate that spastizin interacts with the autophagy related Beclin 1–UVRAG–Rubicon multiprotein complex and is required for autophagosome maturation. In cells lacking spastizin or with mutated forms of the protein, spastizin interaction with Beclin 1 is lost although the formation of the Beclin 1–UVRAG–Rubicon complex can still be observed. However, in these cells we demonstrate an impairment of autophagosome maturation and an accumulation of immature autophagosomes. Autophagy defects with autophagosome accumulation can be observed also in neuronal cells upon spastizin silencing. These results indicate that autophagy is a central process in the pathogenesis of complicated forms of hereditary spastic paraparesis with thin corpus callosum.
PMCID: PMC3784282  PMID: 24030950
spastizin; autophagy; Beclin 1; autophagosome maturation; SPG15
7.  A novel CCM1 mutation associated with multiple cerebral and vertebral cavernous malformations 
BMC Neurology  2014;14:158.
Cerebral cavernous malformations are relatively rare vascular disorders that may affect any part of the central nervous system. This presentation has been associated with heterozygous mutations in CCM1/KRIT1, CCM2/malcavernin and CCM3/PDCD10. We aimed to investigate the genetic defect underlying multiple cerebral and vertebral cavernous malformations in a multigenerational Italian family.
Case presentation
The proband is a 49-year-old man who underwent cerebral MRI in his thirties for persistent haeadache and tingling in his left arm and leg and was diagnosed with multiple supratentorial cavernous angiomas. A right frontal angioma with radiological evidence of a recent bleeding was surgically removed when he was 39 years old and he was thereafter asymptomatic. Magnetic resonance imaging revealed multiple cerebral cavernous malformations in seven members of his familily. Four subjects were asymptomatic. Other family mambers displayed heterogeneous clinical features including seizures and recurrent brain haemorrhages. Sequence analysis in the proband disclosed a novel heterozygous nucleotide substitution (c.263-10A > G) in intron 5 of CCM1. This variant is predicted to create an abnormal acceptor splice site and segregated in affected relatives available for molecular screening. The analysis of CCM1 transcript in proband’s lymphocytes confirmed the partial retention of intron 3 resulting in a premature termination codon.
Our findings demonstrate that c.263-10A > G mutation is associated with cerebral cavernous malformations. A better knowledge of the disease-associated phenotype may lead to an early diagnosis and to an appropriate clinical surveillance in affected patients.
PMCID: PMC4236643  PMID: 25086949
Cerebral Cavernous Malformations; CCM1 protein; Krev interaction trapped 1 protein; Hemangioma; Cavernous; Central Nervous System
8.  Crystal structure of bis­{μ-4-methyl-N′-[3-(oxido­imino)­butan-2-yl­idene]benzene­sulfono­hydrazidato}bis­[(dimethyl sulfoxide-κO)copper(II)] 
In the title compound, [Cu2(C11H13N3O3S)2(C2H6OS)2], the CuII cation is N,N′,O-chelated by a deprotonated hy­droxy­imino-tosyl­hydrazone ligand and coordinated by a dimethyl sulfoxide mol­ecule. One O atom from the adjacent hy­droxy­imino-tosyl­hydrazone ligand bridges the CuII cation, forming the centrosymmetric dimeric complex. The cation is in an overall distorted N2O3 square-pyramidal coordination environment. The methyl­benzene ring is twisted with respect to the hydrazine fragment, with a dihedral angle of 89.54 (9)° between the planes. An intra­molecular C—H⋯O hydrogen bond occurs. In the crystal, mol­ecules are linked by weak C—H⋯O and C—H⋯S inter­actions. Weak π–π stacking is also observed between parallel benzene rings of adjacent mol­ecules, the centroid–centroid distance being 3.9592 (17) Å.
PMCID: PMC4186093  PMID: 25309178
crystal structure; hy­droxy­imino-tosyl­hydrazone derivative; CuII dimer; π–π stacking
9.  Effects of Increased CO2 on Fish Gill and Plasma Proteome 
PLoS ONE  2014;9(7):e102901.
Ocean acidification and warming are both primarily caused by increased levels of atmospheric CO2, and marine organisms are exposed to these two stressors simultaneously. Although the effects of temperature on fish have been investigated over the last century, the long-term effects of moderate CO2 exposure and the combination of both stressors are almost entirely unknown. A proteomics approach was used to assess the adverse physiological and biochemical changes that may occur from the exposure to these two environmental stressors. We analysed gills and blood plasma of Atlantic halibut (Hippoglossus hippoglossus) exposed to temperatures of 12°C (control) and 18°C (impaired growth) in combination with control (400 µatm) or high-CO2 water (1000 µatm) for 14 weeks. The proteomic analysis was performed using two-dimensional gel electrophoresis (2DE) followed by Nanoflow LC-MS/MS using a LTQ-Orbitrap. The high-CO2 treatment induced the up-regulation of immune system-related proteins, as indicated by the up-regulation of the plasma proteins complement component C3 and fibrinogen β chain precursor in both temperature treatments. Changes in gill proteome in the high-CO2 (18°C) group were mostly related to increased energy metabolism proteins (ATP synthase, malate dehydrogenase, malate dehydrogenase thermostable, and fructose-1,6-bisphosphate aldolase), possibly coupled to a higher energy demand. Gills from fish exposed to high-CO2 at both temperature treatments showed changes in proteins associated with increased cellular turnover and apoptosis signalling (annexin 5, eukaryotic translation elongation factor 1γ, receptor for protein kinase C, and putative ribosomal protein S27). This study indicates that moderate CO2-driven acidification, alone and combined with high temperature, can elicit biochemical changes that may affect fish health.
PMCID: PMC4109940  PMID: 25058324
10.  iPSC-Derived Neural Stem Cells Act via Kinase Inhibition to Exert Neuroprotective Effects in Spinal Muscular Atrophy with Respiratory Distress Type 1 
Stem Cell Reports  2014;3(2):297-311.
Spinal muscular atrophy with respiratory distress type 1 (SMARD1) is a motor neuron disease caused by mutations in the IGHMBP2 gene, without a cure. Here, we demonstrate that neural stem cells (NSCs) from human-induced pluripotent stem cells (iPSCs) have therapeutic potential in the context of SMARD1. We show that upon transplantation NSCs can appropriately engraft and differentiate in the spinal cord of SMARD1 animals, ameliorating their phenotype, by protecting their endogenous motor neurons. To evaluate the effect of NSCs in the context of human disease, we generated human SMARD1-iPSCs motor neurons that had a significantly reduced survival and axon length. Notably, the coculture with NSCs ameliorate these disease features, an effect attributable to the production of neurotrophic factors and their dual inhibition of GSK-3 and HGK kinases. Our data support the role of iPSC as SMARD1 disease model and their translational potential for therapies in motor neuron disorders.
Graphical Abstract
•Transplantation of human NSCs ameliorates the SMARD1 phenotype in a disease model•NSCs improve the disease features of SMARD1 motor neurons inhibiting GSK-3 and HGK
Corti and colleagues show that neural stem cells (NSCs) from human-induced pluripotent stem cells (hiPSCs) have therapeutic potential for Spinal Muscular Atrophy with Respiratory Distress Type 1 SMARD1. Transplanted NSCs engraft in the spinal cord of SMARD1 animals, ameliorating their phenotype. SMARD1 hiPSC-derived motor neurons recapitulate disease features that are improved by NSC coculture through GSK-3 and HGK kinase-modulated pathways.
PMCID: PMC4176534  PMID: 25254343
11.  Low PKCa expression within the MRD-HR stratum defines a new subgroup of childhood T-ALL with very poor outcome 
Oncotarget  2014;5(14):5234-5245.
Pediatric T-cell Acute Lymphoblastic Leukemia (T-ALL) outcome has improved in the last decades, yet one patient in every four still relapses. Except treatment response and immunophenotype, few markers are reliably prognostic in pediatric T-ALL patients. Aiming to improve T-ALL risk stratification, we investigated a new candidate biomarker with potential prognostic relevance. A phosphoproteomic screening of 98 pediatric T-ALL samples at diagnosis had been performed using the high-throughput Reverse Phase Protein Arrays technique, which led to the identification of PKCαS657 as an activated protein with a broad variation among T-ALL samples. To evaluate PKCα potential as a prognostic biomarker, PKCα expression was analyzed using RQ-PCR in a cohort of 173 patients, representative of ALL2000-ALLR2006 AIEOP study. A threshold of PKCα expression with the highest discrimination for incidence of relapse was identified. Patients with PKCα down-regulation, compared to patients with PKCα levels above the threshold, presented a markedly increased cumulative incidence of relapse (43.8% vs. 10.9%, P<0.001), as well as a worse 4-year overall survival (66% vs. 87.9%, P=0.002) and event-free survival (53.1% vs. 85.2%, P=0.002). In particular, low PKCα expression identified cases with extremely poor outcome within the high-risk minimal residual disease (MRD) stratum, their incidence of relapse being of 69% vs. 15% in the high PKCα levels group. In a multivariate analysis adjusting for main prognostic features, PKCα proved to be an independent prognostic factor related to incidence of relapse. Very high risk patients within the high-risk MRD stratum, identified by PKCα expression, could be proposed for experimental therapeutic protocols.
PMCID: PMC4170630  PMID: 25026300
Low PKCα expression; Childhood T-cell Acute Lymphoblastic Leukemia; T-ALL prognostic marker
12.  2-Benzoyl-4-chloro­aniline thio­semi­carbazone 
In the title compound, C14H13ClN4S, obtained from a reaction of 2-benzoyl-4-chloro­aniline with thio­semicarbazide in ethanol, the dihedral angle between the aromatic rings is 81.31 (13)°. In the crystal, the mol­ecules are linked by three N—H⋯S hydrogen bonds, forming centrosymmetric rings with set-graph motif R 2 2(8) and R 2 2(18), and resulting in the formation of a two-dimensional network lying parallel to (010).
PMCID: PMC4051094  PMID: 24940260
13.  Epigenetic Silencing of TFPI-2 in Canine Diffuse Large B-Cell Lymphoma 
PLoS ONE  2014;9(4):e92707.
Epigenetic modifications are important early events during carcinogenesis. In particular, hypermethylation of CpG islands in the promoter region of tumor suppressor genes is a well-known mechanism of gene silencing that contributes to cancer development and progression. Tissue factor pathway inhibitor 2 (TFPI-2) is a tumor suppressor involved in invasiveness inhibition. Although TFPI-2 transcriptional silencing, through promoter hypermethylation, has been widely reported in several human malignancies, it has never been explored in lymphoma. In the present study TFPI-2 methylation and gene expression have been investigated in canine Diffuse Large B-cell lymphomas (cDLBCL). The methylation level of 23 CpGs located within the TFPI-2 promoter was investigated by bisulfite-specific PCR and next generation amplicon deep sequencing (GS Junior 454, Roche) in 22 cDLBCLs and 9 controls. For the same specimens, TFPI-2 gene expression was assessed by means of Real-time RT-PCR. Sequence analysis clearly demonstrated that TFPI2 is frequently hypermethylated in cDLBCL. Hypermethylation of the TFPI-2 promoter was found in 77% of DLBCLs (17 out of 22) and in one normal lymph node. Globally, dogs with DLBCL showed a mean methylation level significantly increased compared to controls (p<0.01) and analysis of hypermethylation by site identified 19 loci out of 23 (82%) with mean methylation levels from 2- to 120-fold higher in cDLBCL. Gene expression analysis confirmed a significant down-regulation of TFPI-2 (p<0.05) in DLBCLs compared with normal lymph nodes, suggesting that TFPI-2 hypermethylation negatively regulates its transcription. In addition, a significant positive correlation (p<0.01) was found between TFPI-2 methylation levels and age providing the first indication of age-associated epigenetic modifications in canine DLBCL. To conclude, our findings demonstrated that epigenetic dysregulation of TFPI-2, leading to its reduced expression, is frequently detected in canine DLBCL. In the next future, the aberrant TFPI-2 promoter hypermethylation may be considered in association with prognosis and therapy.
PMCID: PMC3973630  PMID: 24695110
14.  Ancient and Recent Selective Pressures Shaped Genetic Diversity at AIM2-Like Nucleic Acid Sensors 
Genome Biology and Evolution  2014;6(4):830-845.
AIM2-like receptors (ALRs) are a family of nucleic acid sensors essential for innate immune responses against viruses and bacteria. We performed an evolutionary analysis of ALR genes (MNDA, PYHIN1, IFI16, and AIM2) by analyzing inter- and intraspecies diversity. Maximum-likelihood analyses indicated that IFI16 and AIM2 evolved adaptively in primates, with branch-specific selection at the catarrhini lineage for IFI16. Application of a population genetics–phylogenetics approach also allowed identification of positive selection events in the human lineage. Positive selection in primates targeted sites located at the DNA-binding interface in both IFI16 and AIM2. In IFI16, several sites positively selected in primates and in the human lineage were located in the PYD domain, which is involved in protein–protein interaction and is bound by a human cytomegalovirus immune evasion protein. Finally, positive selection was found to target nuclear localization signals in IFI16 and the spacer region separating the two HIN domains. Population genetic analysis in humans revealed that an IFI16 genic region has been a target of long-standing balancing selection, possibly acting on two nonsynonymous polymorphisms located in the spacer region. Data herein indicate that ALRs have been repeatedly targeted by natural selection. The balancing selection region in IFI16 carries a variant with opposite risk effect for distinct autoimmune diseases, suggesting antagonistic pleiotropy. We propose that the underlying scenario is the result of an ancestral and still ongoing host–pathogen arms race and that the maintenance of susceptibility alleles for autoimmune diseases at IFI16 represents an evolutionary trade-off.
PMCID: PMC4007548  PMID: 24682156
AIM2-like receptors; positive selection; long-standing balancing selection; IFI16
15.  An Evolutionary Analysis of Antigen Processing and Presentation across Different Timescales Reveals Pervasive Selection 
PLoS Genetics  2014;10(3):e1004189.
The antigenic repertoire presented by MHC molecules is generated by the antigen processing and presentation (APP) pathway. We analyzed the evolutionary history of 45 genes involved in APP at the inter- and intra-species level. Results showed that 11 genes evolved adaptively in mammals. Several positively selected sites involve positions of fundamental importance to the protein function (e.g. the TAP1 peptide-binding domains, the sugar binding interface of langerin, and the CD1D trafficking signal region). In CYBB, all selected sites cluster in two loops protruding into the endosomal lumen; analysis of missense mutations responsible for chronic granulomatous disease (CGD) showed the action of different selective forces on the very same gene region, as most CGD substitutions involve aminoacid positions that are conserved in all mammals. As for ERAP2, different computational methods indicated that positive selection has driven the recurrent appearance of protein-destabilizing variants during mammalian evolution. Application of a population-genetics phylogenetics approach showed that purifying selection represented a major force acting on some APP components (e.g. immunoproteasome subunits and chaperones) and allowed identification of positive selection events in the human lineage.
We also investigated the evolutionary history of APP genes in human populations by developing a new approach that uses several different tests to identify the selection target, and that integrates low-coverage whole-genome sequencing data with Sanger sequencing. This analysis revealed that 9 APP genes underwent local adaptation in human populations. Most positive selection targets are located within noncoding regions with regulatory function in myeloid cells or act as expression quantitative trait loci. Conversely, balancing selection targeted nonsynonymous variants in TAP1 and CD207 (langerin). Finally, we suggest that selected variants in PSMB10 and CD207 contribute to human phenotypes. Thus, we used evolutionary information to generate experimentally-testable hypotheses and to provide a list of sites to prioritize in follow-up analyses.
Author Summary
Antigen-presenting cells digest intracellular and extracellular proteins and display the resulting antigenic repertoire on cell surface molecules for recognition by T cells. This process initiates cell-mediated immune responses and is essential to detect infections. The antigenic repertoire is generated by the antigen processing and presentation pathway. Because several pathogens evade immune recognition by hampering this process, genes involved in antigen processing and presentation may represent common natural selection targets. Thus, we analyzed the evolutionary history of these genes during mammalian evolution and in the more recent history of human populations. Evolutionary analyses in mammals indicated that positive selection targeted a very high proportion of genes (24%), and revealed that many selected sites affect positions of fundamental importance to the protein function. In humans, we found different signatures of natural selection acting both on regions that are expected to regulate gene expression levels or timing and on coding variants; two human selected polymorphisms may modulate the susceptibility to Crohn's disease and to HIV-1 infection. Therefore, we provide a comprehensive evolutionary analysis of antigen processing and we show that evolutionary studies can provide useful information concerning the location and nature of functional variants, ultimately helping to clarify phenotypic differences between and within species.
PMCID: PMC3967941  PMID: 24675550
16.  Molecular, genetic and stem cell-mediated therapeutic strategies for spinal muscular atrophy (SMA) 
Spinal muscular atrophy (SMA) is an autosomal recessive motor neuron disease. It is the first genetic cause of infant mortality. It is caused by mutations in the survival motor neuron 1 (SMN1) gene, leading to the reduction of SMN protein. The most striking component is the loss of alpha motor neurons in the ventral horn of the spinal cord, resulting in progressive paralysis and eventually premature death. There is no current treatment other than supportive care, although the past decade has seen a striking advancement in understanding of both SMA genetics and molecular mechanisms. A variety of disease modifying interventions are rapidly bridging the translational gap from the laboratory to clinical trials. In this review, we would like to outline the most interesting therapeutic strategies that are currently developing, which are represented by molecular, gene and stem cell-mediated approaches for the treatment of SMA.
PMCID: PMC3930406  PMID: 24400925
spinal muscular atrophy; molecular therapy; antisense oligonucleotides; morpholino; gene therapy; stem-cell therapy; induced pluripotent stem cells
17.  Lipid profiles and inflammatory markers after periodontal treatment in children with congenital heart disease and at risk for atherosclerosis 
Due to the biological associations between periodontal and cardiovascular diseases, as well as the fact that atherosclerosis begins in childhood, behavior based on oral health care and metabolic control from an early age is essential for patients with cardiovascular disease. The aim of this research was to examine the effect of full-mouth scaling and root planing on the reduction of periodontal disease in children with congenital heart disease. In this study, treatments were related to clinical periodontal parameters and also to blood ones, such as lipid profile and inflammatory markers. The patients were divided into two groups: group 1 (n=17), scaling and root planing; and group 2 (n=16), full-mouth scaling and root planing. The results showed a significant improvement in clinical periodontal parameters (P<0.05) in both groups. Considering lipid parameters, total cholesterol, triglycerides, and very-low-density lipoprotein parameters showed significant improvement (P<0.05). There was also an improvement in C-reactive protein (ultrasensitive) in the group treated with scaling and root planing (P<0.05). Fibrinogen and interleukin-6 parameters improved (P<0.05) in both groups. We suggest that both periodontal treatments were effective in children with congenital heart disease, though neither demonstrated superiority.
PMCID: PMC3829709  PMID: 24250224
periodontal disease; heart defects; congenital; lipid metabolism; inflammation mediators; children
18.  Next-generation sequencing reveals DGUOK mutations in adult patients with mitochondrial DNA multiple deletions 
Brain  2012;135(11):3404-3415.
The molecular diagnosis of mitochondrial disorders still remains elusive in a large proportion of patients, but advances in next generation sequencing are significantly improving our chances to detect mutations even in sporadic patients. Syndromes associated with mitochondrial DNA multiple deletions are caused by different molecular defects resulting in a wide spectrum of predominantly adult-onset clinical presentations, ranging from progressive external ophthalmoplegia to multi-systemic disorders of variable severity. The mutations underlying these conditions remain undisclosed in half of the affected subjects. We applied next-generation sequencing of known mitochondrial targets (MitoExome) to probands presenting with adult-onset mitochondrial myopathy and harbouring mitochondrial DNA multiple deletions in skeletal muscle. We identified autosomal recessive mutations in the DGUOK gene (encoding mitochondrial deoxyguanosine kinase), which has previously been associated with an infantile hepatocerebral form of mitochondrial DNA depletion. Mutations in DGUOK occurred in five independent subjects, representing 5.6% of our cohort of patients with mitochondrial DNA multiple deletions, and impaired both muscle DGUOK activity and protein stability. Clinical presentations were variable, including mitochondrial myopathy with or without progressive external ophthalmoplegia, recurrent rhabdomyolysis in a young female who had received a liver transplant at 9 months of age and adult-onset lower motor neuron syndrome with mild cognitive impairment. These findings reinforce the concept that mutations in genes involved in deoxyribonucleotide metabolism can cause diverse clinical phenotypes and suggest that DGUOK should be screened in patients harbouring mitochondrial DNA deletions in skeletal muscle.
PMCID: PMC3501975  PMID: 23043144
DGUOK; mitochondrial DNA instability; autosomal recessive progressive external ophthalmoplegia; multiple mitochondrial DNA deletions
19.  Validation of flow cytometric phospho-STAT5 as a diagnostic tool for juvenile myelomonocytic leukemia 
Blood Cancer Journal  2013;3(11):e160-.
To diagnose juvenile myelomonocytic leukemia (JMML) is sometimes challenging, because around 10% of patients lack molecular abnormalities affecting Ras-MAPK (mitogen-activated protein kinase) pathway and other diseases such as cytomegalovirus infection can mimic clinical signs of JMML. In order to validate a phospho-specific flow cytometry assay assessing phospho-signal transducer and activator of transcription factor 5 (p-STAT5) as a new diagnostic tool for JMML, we examined 22 samples from children with JMML and 47 controls. CD33+/CD34+ cells from 22 patients with JMML showed hyperphosphorylation of STAT5 induced by sub-saturating doses of granulocyte-macrophage colony-stimulating factor (GM-CSF). Using a training set of samples (11 JMML and 23 controls), we identified a threshold for p-STAT5-positive after stimulation with 0.1 ng/ml GM-CSF (17.17%) that discriminates JMML from controls. This threshold was validated in an independent series (11 JMML, 24 controls and 7 cases with diseases other than JMML) where we demonstrated that patients with JMML could be distinguished from other subjects with a sensitivity of 91% (confidence interval (CI) 59–100%) and a specificity of 87% (CI 70–96%). Positive and negative predictive values were 71% (CI 42–92%) and 96% (CI 82–100%), respectively. In conclusion, flow cytometric p-STAT5 profiling is a reliable diagnostic tool for identifying patients with JMML and can contribute to consistency of current diagnostic criteria.
PMCID: PMC3880439  PMID: 24241400
juvenile myelomonocytic leukemia; phospho-specific flow cytometry; phospho-STAT5; GM-CSF
20.  Optic Neuritis as Isolated Manifestation of Leptomeningeal Carcinomatosis: A Case Report and Systematic Review of Ocular Manifestations of Neoplastic Meningitis 
Introduction. Leptomeningeal carcinomatosis occurs in about 5% of cancer patients. Ocular involvement is a common clinical manifestation and often the presenting clinical feature. Materials and Methods. We report the case of a 52-year old lady with optic neuritis as isolated manifestation of neoplastic meningitis and a review of ocular involvement in neoplastic meningitis. Ocular symptoms were the presenting clinical feature in 34 patients (83%) out of 41 included in our review, the unique manifestation of meningeal carcinomatosis in 3 patients (7%). Visual loss was the presenting clinical manifestation in 17 patients (50%) and was the most common ocular symptom (70%). Other ocular signs were diplopia, ptosis, papilledema, anisocoria, exophthalmos, orbital pain, scotomas, hemianopsia, and nystagmus. Associated clinical symptoms were headache, altered consciousness, meningism, limb weakness, ataxia, dizziness, seizures, and other cranial nerves involvement. All patients except five underwent CSF examination which was normal in 1 patient, pleocytosis was found in 11 patients, increased protein levels were observed in 16 patients, and decreased glucose levels were found in 8 patients. Cytology was positive in 29 patients (76%). Conclusion. Meningeal carcinomatosis should be considered in patients with ocular symptoms even in the absence of other suggestive clinical symptoms.
PMCID: PMC3816070  PMID: 24223306
21.  Growing Evidence about the Relationship between Vessel Dissection and Scuba Diving 
Case Reports in Neurology  2013;5(3):155-161.
Carotid and vertebral artery dissection are relatively frequent and risky conditions. In the last decade, different patients with extracranial (and in 1 case also intracranial) dissections associated with the practice of scuba diving were reported. The connection between the two conditions has not been fully explained so far. In the present article, we report the case of a patient presenting with Claude Bernard-Horner syndrome and homolateral XII cranial nerve palsy, manifesting a few days after diving in the cold water of a lake. The patient ended up having internal carotid artery dissection associated with the formation of a pseudoaneurysm. Here, we offer a summary of all cases reported in the literature about scuba diving and arterial dissection, and provide a critical discussion about which scuba diving-related factors can trigger the dissection of cervical vessels.
PMCID: PMC3806682  PMID: 24163671
Internal carotid dissection; Scuba diving; Claude Bernard-Horner syndrome; XII cranial nerve
22.  Minimally invasive transplantation of iPSC-derived ALDHhiSSCloVLA4+ neural stem cells effectively improves the phenotype of an amyotrophic lateral sclerosis model 
Human Molecular Genetics  2013;23(2):342-354.
Amyotrophic lateral sclerosis (ALS) is a fatal neurological disease characterized by the degeneration of motor neurons. Currently, there is no effective therapy for ALS. Stem cell transplantation is a potential therapeutic strategy for ALS, and the reprogramming of adult somatic cells into induced pluripotent stem cells (iPSCs) represents a novel cell source. In this study, we isolated a specific neural stem cell (NSC) population from human iPSCs based on high aldehyde dehydrogenase activity, low side scatter and integrin VLA4 positivity. We assessed the therapeutic effects of these NSCs on the phenotype of ALS mice after intrathecal or intravenous injections. Transplanted NSCs migrated and engrafted into the central nervous system via both routes of injection. Compared with control ALS, treated ALS mice exhibited improved neuromuscular function and motor unit pathology and significantly increased life span, in particular with the systemic administration of NSCs (15%). These positive effects are linked to multiple mechanisms, including production of neurotrophic factors and reduction of micro- and macrogliosis. NSCs induced a decrease in astrocyte number through the activation of the vanilloid receptor TRPV1. We conclude that minimally invasive injections of iPSC-derived NSCs can exert a therapeutic effect in ALS. This study contributes to advancements in iPSC-mediated approaches for treating ALS and other neurodegenerative diseases.
PMCID: PMC3869354  PMID: 24006477
23.  An evolutionary history of the selectin gene cluster in humans 
Heredity  2012;109(2):117-126.
Molecules involved in leukocyte trafficking have a central role in the development of inflammatory and immune responses. We performed FST analysis of the selectin cluster, as well as of SELPLG, ICAM1 and VCAM1. Peaks of significantly high population genetic differentiation were restricted to two regions in SELP and one in SELPLG. Resequencing data indicated that the region covering SELP exons 11–13 displays high nucleotide diversity in Africans and Europeans (CEU), and a high level of within-species diversity compared with inter-specific divergence. Analysis of inferred haplotypes revealed a complex phylogeny with two deeply separated clades that coalesce at ∼3.5 million years (MY) plus a minor clade with a TMRCA (time to the most recent common ancestor) of ∼2.2 MY. A splicing assay indicated no haplotype-specific effect on SELP exon 14 inclusion. These data are consistent with a model of multiallelic balancing selection; single-nucleotide polymorphism analysis indicated that the Val640Leu variant represents a likely selection target. In populations of Asian ancestry a distinct haplotype, possibly carrying regulatory variants, has been driven to high frequency by positive selection. No deviation from neutrality was observed for the SELPLG region. Resequencing of SELP in chimpanzees revealed a haplotype phylogeny with extremely deep basal branches, suggesting either long-standing balancing selection or ancestral population structure. Thus, SELP has experienced a complex selective history, possibly as a result of local adaptation. Variants in the gene have been associated with autoimmune and cardiovascular diseases. Association studies would benefit from both taking the complex SELP haplotype structure into account and from analysis of possible regulatory variants in the gene.
PMCID: PMC3400748  PMID: 22549518
selectin gene cluster; SELP; balancing selection; local adaptation
24.  1-(5-Bromo-2-oxoindolin-3-yl­idene)-4-phenyl­thio­semicarbazide 
In the title compound, C15H11BrN4OS, the least-squares plane through the 5-bromo­isatin fragment forms a dihedral angle of 13.63 (14)° with the phenyl ring. The mol­ecular conformation features intra­molecular N—H⋯N and N—H⋯O hydrogen bonds. In the crystal, mol­ecules are connected via pairs of N—H⋯O inter­actions into centrosymmetric dimers. Additionally, π–π stacking inter­actions link mol­ecules into chains parallel to the a axis with short C⋯C distances being observed between the phenyl and thio­carbonyl [3.236 (8) Å] groups and between the thio­carbonyl and carbonyl [3.351 (4) Å] groups of stacked mol­ecules.
PMCID: PMC3793820  PMID: 24109407
25.  1-(5-Bromo-2-oxoindolin-3-yl­idene)thio­semicarbazone 
The title mol­ecule, C9H7BrN4OS, is essentially planar [r.m.s. deviation = 0.066 (2) Å], the maximum deviation from the mean plane through the non-H atoms being 0.190 (3) Å for the terminal amine N atom. In the crystal, mol­ecules are linked through N—H⋯O and N—H⋯S inter­actions, generating infinite chains along the b-axis direction. In turn, the chains are stacked along the a axis via π–π inter­actions [centroid–centroid distance = 3.470 (2) Å] and further connected by N—H⋯Br inter­actions into a three-dimensional network. An intra­molecular N—H⋯O hydrogen bond is also observed.
PMCID: PMC3793752  PMID: 24109339

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