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1.  The Major Cow Milk Allergen Bos d 5 Manipulates T-Helper Cells Depending on Its Load with Siderophore-Bound Iron 
PLoS ONE  2014;9(8):e104803.
The mechanisms of allergic sensitization to milk are still elusive. The major allergen Bos d 5 belongs to the lipocalin-family and thus is able to transport numerous ligands. In this study we investigated its ability to bind to iron-siderophore complexes and tested the immune-modulatory properties of Bos d 5 in either forms. Structural and in silico docking analysis of Bos d 5 revealed that Bos d 5 is able to bind to iron via catechol-based flavonoids (quercetin, myricetin, luteolin) that act as siderophores as confirmed by spectral-analysis and iron staining. Calculated dissociation constants of docking analyses were below 1 µM by virtual addition of iron. When incubated with human peripheral blood mononuclear cells (PBMCs), only the apo-form of Bos d 5 led to an increase of CD4+positive cells and significantly elevated IL13 and IFNγ-levels. In contrast, holo-Bos d 5 decreased numbers of CD4 expressing cells and induced apoptosis. Taken together, our data give evidence that Bos d 5 is capable of binding iron via siderophores. Moreover, our data support for the first time the notion that the form of application (apo- or holo-form) is decisive for the subsequent immune response. The apo-form promotes Th2 cells and inflammation, whereas the holo-form appears to be immunosuppressive.
PMCID: PMC4130594  PMID: 25117976
2.  Navicular index for differentiation of flatfoot from normal foot 
International Orthopaedics  2013;37(6):1107-1112.
The height of navicular bone from the floor is in proportion with the height of longitudinal arch of the foot. The study was conducted to evaluate correlation of navicular bone height with most often used angles, heel valgus and a foot print in order to simplify the procedure for the diagnosis of flatfoot.
A total of 218 operated children (436 feet) because of flexible flatfoot were evaluated clinically and radiologically. Meary angle, lateral talonavicular angle, talocalcaneal angle, calcaneal pitch, heel valgus and arch index (Staheli) were evaluated pre-operatively and postoperatively. In 121 (242 feet) chosen children (age eight to 15) with all clinical values and pre-operative angles corresponding flatfoot, all postoperatively measured values were within the normal range. We got the navicular index by dividing length of longitudinal arch with navicular height. Values of navicular index were then compared with pre-operatively and postoperatively measured values. Pearson correlation and ROC test were used for statistical analysis.
Values of the navicular index for flatfeet were in the interval from 4.75 to 31.2 (median 8.98), and for normal-arched feet 3.58–22.6 (median 5.48). Pearson correlation of arch index and measured parameters were significant in majority, and degree according to Colton was good. Area under the ROC curve was 0.861 (p = 0.0001). The cut-off value with 86 % sensitivity and 75 % specificity was 6.7407.
Navicular index can be used reliably, without measures of the other parameters, to differentiate flatfoot from normal-arched foot. Therefore, the navicular index has an ability to distinguish between the flatfoot and normal-arched foot.
PMCID: PMC3664154  PMID: 23580031
3.  Bet v 1 from Birch Pollen Is a Lipocalin-like Protein Acting as Allergen Only When Devoid of Iron by Promoting Th2 Lymphocytes* 
The Journal of Biological Chemistry  2014;289(25):17416-17421.
Background: The reason of allergic sensitization to proteins like Bet v 1 is unknown.
Results: Bet v 1 binds iron via catechol-based siderophores. It can modulate human immune cells toward Th2 when not carrying iron.
Conclusion: Bet v 1 modulates immune cells toward Th2 when being devoid of iron.
Significance: We provide the functional basis under which circumstances Bet v 1 becomes an allergen.
It is hypothesized that allergens are at the borderline of self and non-self and, through as yet elusive circumstances, mount a Th2 response for allergic sensitization. The major birch pollen allergen Bet v 1 is considered the prototype for the PR-10 protein family causing respiratory allergy. Here, we give structural evidence that Bet v 1 is a lipocalin-like protein with a striking structural resemblance to human lipocalin 2. Lipocalin 2 is highly expressed in the lung where it exerts immunoregulatory functions dependent on being loaded with siderophore-bound iron (holo-form) or not (apo-form). We demonstrate that similar to lipocalin 2, Bet v 1 is capable of binding iron via catechol-based siderophores. Thereby, calculated Kd values of 66 nm surpassed affinities to known ligands nearly by a power of 10. Moreover, we give functional evidence of the immunomodulatory capacity of Bet v 1 being dependent on its iron-loaded state. When incubated to human immune cells, only the apo-form of Bet v 1, but not the holo-form, was able to promote Th2 cells secreting IL13. These results provide for the first time a functional understanding on the allergenicity of Bet v 1 and a basis for future allergen immunotherapies counteracting Th2 immune responses on a molecular basis.
PMCID: PMC4067174  PMID: 24798325
Allergen; Allergy; Immunosuppression; Iron; Mucosal Immunology; Siderophore; Bet v 1; Th2 Skewing; Allergic Sensitization; Apo- and Holo-form
4.  Differential Expression of Intracellular and Extracellular CB2 Cannabinoid Receptor Protein by Human Peripheral Blood Leukocytes 
mRNA encoding for the CB2 cannabinoid receptor is expressed by many subsets of human peripheral blood leukocytes (PBL), but little is known about the resulting protein expression and function. Employing clones from the A549 and 293T cell lines that were constructed to express both full-length human CB2 and GFP, we developed a flow cytometry assay for characterizing CB2 protein expression. A monoclonal antibody directed against human CB2 selectively stained the surface of transduced but not parental cell lines. When cells were fixed and permeabilized, imaging flow cytometry identified large stores of intracellular protein. Total cellular staining for CB2 corresponded closely with the level of GFP expression. When exposed to Δ-9-tetrahydrocannabinol, CB2-expressing cells internalized cell surface CB2 receptors in a time- and dose-dependent manner. Applying these approaches to human PBL, CB2 protein was identified on the surface of human B cells but not on T cells or monocytes. In contrast, when PBL were fixed and permeabilized, intracellular CB2 expression was readily detected in all three subsets by both conventional and imaging flow cytometry. Similar to the protein expression pattern observed in fixed and permeabilized PBL, purified B cells, T cells, and monocytes expressed relatively equal levels of CB2 mRNA by quantitative real-time RT-PCR. Our findings confirm that human PBL express CB2 protein but that its distribution is predominantly intracellular with only B cells expressing CB2 protein at the extracellular membrane. The differential role of intracellular and extracellular CB2 receptors in mediating ligand signaling and immune function remains to be determined.
PMCID: PMC3587044  PMID: 23299999
Cannabinoids; Cannabinoid Receptor CB2; G-protein-Coupled Receptors; Intracellular Membrane Receptors; Leukocytes; Imaging Flow Cytometry; Tetrahydrocannabinol
5.  Age-related changes in Arc transcription and DNA methylation within the hippocampus 
Neurobiology of aging  2010;32(12):2198-2210.
The transcription of genes that support memory processes are likely to be impacted by the normal aging process. Because Arc is necessary for memory consolidation and enduring synaptic plasticity, we examined Arc transcription within the aged hippocampus. Here, we report that Arc transcription is reduced within the aged hippocampus compared to the adult hippocampus during both “off line” periods of rest, and following spatial behavior. This reduction is observed within ensembles of CA1 “place cells”, which make less mRNA per cell, and in the dentate gyrus (DG) where fewer granule cells are activated by behavior. In addition, we present data suggesting that aberrant changes in methylation of the Arc gene may be responsible for age-related decreases in Arc transcription within CA1 and the DG. Given that Arc is necessary for normal memory function, these subregion-specific epigenetic and transcriptional changes may result in less efficient memory storage and retrieval during aging.
PMCID: PMC2888808  PMID: 20189687
6.  Centrosome misorientation mediates slowing of the cell cycle under limited nutrient conditions in Drosophila male germline stem cells 
Molecular Biology of the Cell  2012;23(8):1524-1532.
A novel mechanism is found by which Drosophila male germline stem cells (GSCs) slow their cell cycle under limited nutrient conditions. Upon culturing in poor media, GSCs misorient their centrosomes with respect to the stem cell niche, activating the centrosome orientation checkpoint and leading to slowdown of the cell cycle.
Drosophila male germline stem cells (GSCs) divide asymmetrically, balancing self-renewal and differentiation. Although asymmetric stem cell division balances between self-renewal and differentiation, it does not dictate how frequently differentiating cells must be produced. In male GSCs, asymmetric GSC division is achieved by stereotyped positioning of the centrosome with respect to the stem cell niche. Recently we showed that the centrosome orientation checkpoint monitors the correct centrosome orientation to ensure an asymmetric outcome of the GSC division. When GSC centrosomes are not correctly oriented with respect to the niche, GSC cell cycle is arrested/delayed until the correct centrosome orientation is reacquired. Here we show that induction of centrosome misorientation upon culture in poor nutrient conditions mediates slowing of GSC cell proliferation via activation of the centrosome orientation checkpoint. Consistently, inactivation of the centrosome orientation checkpoint leads to lack of cell cycle slowdown even under poor nutrient conditions. We propose that centrosome misorientation serves as a mediator that transduces nutrient information into stem cell proliferation, providing a previously unappreciated mechanism of stem cell regulation in response to nutrient conditions.
PMCID: PMC3327310  PMID: 22357619
7.  Predictors of disease severity in ulcerative colitis patients from Southwestern Ontario 
AIM: To understand the demographic characteristics of patients in Southwestern Ontario, Canada with ulcerative colitis (UC) in order to predict disease severity.
METHODS: Records from 1996 to 2001 were examined to create a database of UC patients seen in the London Health Sciences Centre South Street Hospital Inflammatory Bowel Disease Clinic. To be included, patients’ charts were required to have information of their disease presentation and a minimum of 5 years of follow-up. Charts were reviewed using standardized data collection forms. Disease severity was generated during the chart review process, and non-endoscopic Mayo Score criteria were collected into a composite.
RESULTS: One hundred and two consecutive patients’ data were entered into the database. Demographic analyses revealed that 51% of the patients were male, the mean age at diagnosis was 39 years, 13.7% had a first degree relative with inflammatory bowel disease (IBD), 61.8% were nonsmokers and 24.5% were ex-smokers. In 22.5% of patients the disease was limited to the rectum, in 21.6% disease was limited to the sigmoid colon, in 22.5% disease was limited to the left colon, and 32.4% of patients had pancolitis. Standard multiple regression analysis which regressed a composite of physician global assessment of disease severity, average number of bowel movements, and average amount of blood in bowel movements on year of diagnosis and age at time of diagnosis was significant, R2 = 0.306, F (7, 74) = 4.66, P < 0.01. Delay from symptoms to diagnosis of UC, gender, family history of IBD, smoking status and disease severity at the time of diagnosis didnot significantly predict the composite measure.
CONCLUSION: UC severity is associated with younger age at diagnosis and year of diagnosis in a longitudinal cohort of UC patients, and may identify prognostic UC indicators.
PMCID: PMC2806562  PMID: 20066743
Demographic; Disease severity; Prognosis; Ulcerative colitis
8.  Value of PCR for evaluating occurrence of parasitemia in immunocompromised patients with cerebral and extracerebral toxoplasmosis. 
Journal of Clinical Microbiology  1994;32(11):2813-2819.
PCR was used to evaluate the occurrence of Toxoplasma gondii parasitemia by detection of the B1 gene in blood samples in two groups of immunosuppressed patients (148 subjects) suspected of having cerebral or extracerebral infection, respectively. Group I consisted of 52 patients with AIDS with suspected cerebral toxoplasmosis. The diagnosis was clinically proven in 15 cases. Parasitemia was detected by PCR in only two of these patients (13.3%), both showing evidence of disseminated infection. Group II consisted of 96 immunocompromised patients, either with AIDS or receiving iatrogenic immunosuppressive therapy. Of these patients, 65 (34 with AIDS and 31 others) showed abnormalities only in chest radiography and were first screened for the presence of Toxoplasma DNA in bronchoalveolar lavage fluid. Blood was then analyzed when the parasite was detected in the bronchoalveolar lavage fluid. The remaining 31 subjects (22 with AIDS and 9 others) were suspected of having extracerebral, pulmonary, or disseminated toxoplasmosis, and blood was studied directly in these cases. Among the nine patients with clinically diagnosed extracerebral infection in group II, the parasite was detected by PCR in the blood of five patients (55.5%), all having pulmonary toxoplasmosis. If all patients with clinical manifestations of extracerebral toxoplasmosis (from both groups) who had not received antitoxoplasma therapy when the samples were collected are considered, PCR detected parasitemia in seven of the nine cases (77.8%). The present study indicates that examination of blood by PCR may be valuable in cases of extracerebral toxoplasmosis because of the disseminated nature of the disease. Since most cases of cerebral toxoplasmosis result from the local reactivation of latent brain cysts, detection of parasitemia by PCR is useful only in cases associated with severe cerebral infection or dissemination of this disease.
PMCID: PMC264163  PMID: 7852576
9.  Cognitive Effects of Hormone Therapy in Men With Prostate Cancer 
Cancer  2008;113(5):1097-1106.
Men who receive androgen-deprivation therapy (ADT) for prostate cancer experience several side effects from this treatment. A few recent studies have examined the cognitive implications of ADT and how they impact a patient’s treatment decision-making, occupational pursuits, and quality of life. For this report, the authors explored possible mechanisms for this association, reviewed research in animal studies and aging men, and examined the growing literature focused on the relation between ADT and cognitive functioning in patients with prostate cancer.
A systematic literature search was conducted using the PubMed and Information Sciences Institute Web of Knowledge-Web of Science databases to identify relevant studies that investigated the relation between ADT in men with prostate cancer and its cognitive effects.
Testosterone and its derivatives may have an impact on cognition through several mechanisms in the brain, as supported by studies of animals and in aging men. Studies that researched the impact of ADT on cognition in patients with prostate cancer patients were designed relatively well but suffered from small sample sizes. Between 47% and 69% of men on ADT declined in at least 1 cognitive area, most commonly in visuospatial abilities and executive functioning. Some studies reported contradictory results with increased functioning in verbal memory.
There is a strong argument that androgen-ablation therapy is linked to subtle but significant cognitive declines in men with prostate cancer. The authors believe that clinicians should become aware of this correlation as the use of ADT increases and should inform and monitor patients for this possible side effect of treatment.
PMCID: PMC4333639  PMID: 18666210
altered cognitive function; androgen ablation; dihydrotestosterone; estradiol; prostate cancer; quality of life; testosterone
10.  Rodent Model of Infant Attachment Learning and Stress 
Developmental psychobiology  2010;52(7):651-660.
Here we review the neurobiology of infant odor learning in rats, and discuss the unique role of the stress hormone corticosterone (CORT) in the learning necessary for the developing rat. During the first 9 postnatal (PN) days, infants readily learn odor preferences, while aversion and fear learning are attenuated. Such restricted learning may ensure that pups only approach their mother. This sensitive period of preference learning overlaps with the stress hyporesponsive period (SHRP, PN4–14) when pups have a reduced CORT response to most stressors. Neural underpinnings responsible for sensitive-period learning include increased activity within the olfactory bulb and piriform “olfactory” cortex due to heightened release of norepinephrine from the locus coeruleus. After PN10 and with the decline of the SHRP, stress-induced CORT release permits amygdala activation and facilitates learned odor aversions and fear. Remarkably, odor preference and attenuated fear learning can be reestablished in PN10–15 pups if the mother is present, an effect due to her ability to suppress pups’ CORT and amygdala activity. Together, these data indicate that functional changes in infant learning are modified by a unique interaction between the developing CORT system, the amygdala, and maternal presence, providing a learning system that becomes more flexible as pups mature.
PMCID: PMC4334117  PMID: 20730787
mother–infant interactions; olfactory bulb; norepinephrine; attachment; imprinting; locus coeruleus; amygdala; learning; classical conditioning; corticosterone; stress; fear
11.  Assessment of MS/MS Search Algorithms with Parent-Protein Profiling 
Journal of Proteome Research  2014;13(4):1823-1832.
Peptide mass spectrometry relies crucially on algorithms that match peptides to spectra. We describe a method to evaluate the accuracy of these algorithms based on the masses of parent proteins before trypsin endoprotease digestion. Measurement of conformance to parent proteins provides a score for comparison of the performances of different algorithms as well as alternative parameter settings for a given algorithm. Tracking of conformance scores for spectrum matches to proteins with progressively lower expression levels revealed that conformance scores are not uniform within data sets but are significantly lower for less abundant proteins. Similarly peptides with lower algorithm peptide-spectrum match scores have lower conformance. Although peptide mass spectrometry data is typically filtered through decoy analysis to ensure a low false discovery rate, this analysis confirms that the filtered data should not be considered as having a uniform confidence. The analysis suggests that use of different algorithms and multiple standardized parameter settings of these algorithms can increase significantly the numbers of peptides identified. This data set can be used as a resource for future algorithm assessment.
PMCID: PMC3993968  PMID: 24533481
peptide mass spectrometry; trypsin; OMSSA; SEQUEST; Mascot; algorithm parameter sets; parent-protein conformance; decoy analysis
12.  HTS navigator: freely accessible cheminformatics software for analyzing high-throughput screening data 
Bioinformatics  2013;30(4):588-589.
Summary: We report on the development of the high-throughput screening (HTS) Navigator software to analyze and visualize the results of HTS of chemical libraries. The HTS Navigator processes output files from different plate readers' formats, computes the overall HTS matrix, automatically detects hits and has different types of baseline navigation and correction features. The software incorporates advanced cheminformatics capabilities such as chemical structure storage and visualization, fast similarity search and chemical neighborhood analysis for retrieved hits. The software is freely available for academic laboratories.
Availability and implementation:
Supplementary information: Supplementary data are available at Bioinformatics online.
PMCID: PMC3928525  PMID: 24376084
13.  Ganglioside GD2 as a Therapeutic Target for Antibody-Mediated Therapy in Osteosarcoma 
Cancer  2013;120(4):548-554.
Survival outcomes for patients with osteosarcoma have remained stagnant over the past three decades. Targeting of ganglioside GD2, a glycosphingolipid on the cell surface of some tumors, with immunotherapy has resulted in improved outcomes for patients with neuroblastoma. The expression pattern of GD2 was examined in osteosarcoma.
Immunohistochemistry was performed on osteosarcoma samples from patients at the time of initial biopsy, definitive surgery, and recurrence. The intensity and location of staining were scored. Cell-based ELISA was performed on osteosarcoma cell lines to quantitate the level of GD2 expression.
Forty-four osteosarcoma samples were evaluated by immunohistochemistry, including 8 samples from the initial biopsy, 28 samples from the definitive surgery, and 8 samples from the time of recurrence. GD2 was expressed on all 44 osteosarcoma samples. Osteosarcoma tissue obtained at the time of recurrence showed higher intensity of staining compared to samples obtained at initial biopsy and definitive surgery (p=0.016). The majority of osteosarcoma cell lines expressed GD2 at higher levels than the neuroblastoma cell line BE(2)-C.
Ganglioside GD2 is highly expressed on osteosarcomas. Clinical trials are needed to assess the efficacy of targeting GD2 in patients with osteosarcoma.
PMCID: PMC3946333  PMID: 24166473
osteosarcoma; ganglioside GD2; immunotherapy; antibody
14.  Molecular Control of δ-Opioid Receptor Signaling 
Nature  2014;506(7487):191-196.
Opioids represent widely prescribed and abused medications, although their signal transduction mechanisms are not well understood. Here we present the 1.8Å high-resolution crystal structure of the human δ-opioid receptor (δ-OR), revealing the presence and fundamental role of a sodium ion mediating allosteric control of receptor functional selectivity and constitutive activity. The distinctive δ-OR sodium ion site architecture is centrally located in a polar interaction network in the 7-transmembrane bundle core, with the sodium ion stabilizing a reduced agonist affinity state, and thereby modulating signal transduction. Site-directed mutagenesis and functional studies reveal that changing the allosteric sodium site residue Asn131 to alanine or valine augments constitutive arrestin-ergic signaling. Asp95Ala, Asn310Ala, and Asn314Ala mutations transform classical δ-opioid antagonists like naltrindole into potent β-arrestin-biased agonists. The data establish the molecular basis for allosteric sodium ion control in opioid signaling, revealing that sodium-coordinating residues act as “efficacy-switches” at a prototypic G protein-coupled receptor.
PMCID: PMC3931418  PMID: 24413399
human opioid receptor; sodium regulation; allostery; functional selectivity; GPCR signaling; constitutive activity; arrestin
15.  Identification of distinct basal and luminal subtypes of muscle-invasive bladder cancer with different sensitivities to frontline chemotherapy 
Cancer cell  2014;25(2):152-165.
Muscle-invasive bladder cancers (MIBCs) are biologically heterogeneous and have widely variable clinical outcomes and responses to conventional chemotherapy. We discovered 3 molecular subtypes of MIBC that resembled established molecular subtypes of breast cancer. Basal MIBCs shared biomarkers with basal breast cancers and were characterized by p63 activation, squamous differentiation, and more aggressive disease at presentation. Luminal MIBCs contained features of active PPARγ and estrogen receptor (ER) transcription and were enriched with activating FGFR3 mutations and potentially FGFR inhibitor sensitivity. p53-like MIBCs were consistently resistant to neoadjuvant MVAC chemotherapy, and all chemoresistant tumors adopted a p53-like phenotype after therapy. Our observations have important implications for prognostication, the future clinical development of targeted agents, and disease management with conventional chemotherapy.
PMCID: PMC4011497  PMID: 24525232
16.  Mechanistic Studies of Gold and Palladium Cooperative Dual-Catalytic Cross-Coupling Systems 
ACS catalysis  2014;4(2):622-629.
Double-label crossover, modified-substrate, and catalyst comparison experiments in the gold and palladium dual-catalytic rearrangement/cross-coupling of allenoates were performed in order to probe the mechanism of this reaction. The results are consistent with a cooperative catalysis mechanism whereby 1) gold activates the substrate prior to oxidative addition by palladium, 2) gold acts as a carbophilic rather than oxophilic Lewis acid, 3) competing olefin isomerization is avoided, 4) gold participates beyond the first turnover and therefore does not serve simply to generate the active palladium catalyst, and 5) single-electron transfer is not involved. These experiments further demonstrate that the cooperativity of both gold and palladium in the reaction is essential because significantly lower to zero conversion is achieved with either metal alone in comparison studies that examined multiple potential gold, palladium, and silver catalysts and precatalysts. Notably, employment of the optimized cocatalysts, PPh3AuOTf and Pd2dba3, separately (i.e., only Au or only Pd) results in zero conversion to product at all monitored time points compared to quantitative conversion to product when both are present in cocatalytic reactions.
PMCID: PMC3991925  PMID: 24757581
Gold and Palladium; Cooperative catalysis; Dual catalysis; Cross-coupling; Crossover; Mechanism; Intermediates
18.  Acute Mastoiditis in the Pneumococcal Conjugate Vaccine Era 
Following the introduction of the 7- and 13-valent pneumococcal conjugate vaccines, we observed an inverse relationship between the increasing rate of immunized children and the proportion of middle ear fluid cultures collected during acute mastoiditis episodes that tested positive for Streptococcus pneumoniae among a subset of children 0 to 6 years old who had initially presented with severe acute otitis media and had bacterial cultures collected during tympanocentesis or from spontaneous otorrhea.
PMCID: PMC4135914  PMID: 24920600
19.  Cluster of Infections Caused by Methicillin-Resistant Staphylococcus pseudintermedius in Humans in a Tertiary Hospital 
Journal of Clinical Microbiology  2014;52(8):3118-3120.
The dog-associated Staphylococcus pseudintermedius is a rare pathogen in humans. Here we describe a cluster of infections caused by the methicillin-resistant S. pseudintermedius clone ST71-J-t02-II–III. It involved four elderly patients at a tertiary hospital. Three patients had wound infections, and the strain had a tendency to cause bullous skin lesions.
PMCID: PMC4136194  PMID: 24871217
20.  Parental Responsiveness Moderates the Association Between Early-life Stress and Reduced Telomere Length 
Development and psychopathology  2013;25(3):577-585.
Early-life stress, such as maltreatment, institutionalization, and exposure to violence, is associated with accelerated telomere shortening. Telomere shortening may thus represent a biomarker of early adversity. Previous studies have suggested that responsive parenting may protect children from the negative biological and behavioral consequences of early adversity. This study examined the role of parental responsiveness in buffering children from telomere shortening following experiences of early-life stress. We found that high-risk children had significantly shorter telomeres than low-risk children, controlling for household income, birth weight, gender, and minority status. Further, parental responsiveness moderated the association between risk and telomere length, with more responsive parenting associated with longer telomeres only among high-risk children. These findings suggest that responsive parenting may have protective benefits on telomere shortening for young children exposed to early-life stress. Accordingly, this study has important implications for early parenting interventions.
PMCID: PMC4312590  PMID: 23527512
early-life stress; telomeres; child maltreatment; maternal sensitivity; parental behavior
21.  In-vivo analysis of epicutaneous pressure distribution beneath a femoral tourniquet – an observational study 
Compression of the tissue beneath tourniquets used in limb surgery is associated with varying degrees of soft tissue damage. The interaction between fluids and applied pressure seems to play an important role in the appearance of skin lesions. The extent of the transfer of force between the tourniquet and the skin, however, has yet to be studied. The aim of the present study was to quantify in-vivo the transfer of pressure between a tourniquet and the skin of the thigh.
Pressure under the tourniquet was measured using sensors in 25 consecutive patients over the course of elective surgical procedures. Linear mixed modeling was used to assess the homogeneity of the distribution of pressure around the circumference of the limb, variation in pressure values over time, and the influence of limb circumference and the Body-Mass-Index (BMI) on pressure transfer.
Mean pressure on the skin was significantly lower than the inner pressure of the cuff (5.95%, 20.5 ± 9.36 mmHg, p < 0.01). There was a discrete, but significant (p < 0.001) increase in pressure within the first twenty minutes after inflation. Sensors located in the area of overlap of the cuff registered significantly higher pressure values (p < 0.01). BMI and leg circumference had no influence on the transfer of pressure to the surface of the skin (p = 0.88 and p = 0.51).
Pressure transfer around the circumference of the limb was distributed inhomogeneously. The measurement series revealed a global pressure drop compared to the initial pressure of the cuff. No relationship could be demonstrated between the pressure transferred to the skin and the BMI or limb circumference.
PMCID: PMC4327976  PMID: 25637090
22.  Rad50-CARD9 interactions link cytosolic DNA sensing to IL-1β production 
Nature immunology  2014;15(6):538-545.
Double-stranded DNA (dsDNA) in the cytoplasm triggers interleukin-1β (IL-1β) production as an anti-viral host response, and deregulation of the pathways involved can promote inflammatory disease. Here we report a direct cytosolic interaction between the DNA-damage sensor Rad50 and the innate immune adapter CARD9. Dendritic cell transfection with dsDNA or infection with a DNA virus induces the formation of dsDNA-Rad50-CARD9 signaling complexes for NF-κB activation and pro-IL-1β generation. Primary cells conditionally deficient for Rad50 or lacking CARD9 consequently exhibit defective DNA-induced IL-1β production, and Card9−/− mice have impaired inflammatory responses upon DNA virus infection in vivo. These results define a cytosolic DNA recognition pathway for inflammation and a physical and functional connection between a conserved DNA-damage sensor and the innate immune response to pathogens.
PMCID: PMC4309842  PMID: 24777530
23.  Prolonged release melatonin for improving sleep in totally blind subjects: a pilot placebo-controlled multicenter trial 
Melatonin, secreted by the pineal gland during the night phase, is a regulator of the biological clock and sleep tendency. Totally blind subjects frequently report severe, periodic sleep problems, with 50%–75% of cases displaying non-24-hour sleep–wake disorder (N24HSWD) due to inability to synchronize with the environmental day–night cycle. Melatonin immediate-release preparations are reportedly effective in N24HSWD. Here, we studied the efficacy and safety of prolonged-release melatonin (PRM), a registered drug for insomnia, for sleep disorders in totally blind subjects living in normal social environments. The primary endpoint was demonstration of clinically meaningful effects on sleep duration (upper confidence interval [CI] limit >20 minutes whether significant or not) to allow early decision-making on further drug development in this indication.
Trial registration registry – NCT00972075.
In a randomized, double-blind, placebo-controlled proof-of-principle study, 13 totally blind subjects had 2 weeks’ placebo run-in, 6 weeks’ randomized (1:1) PRM (Circadin®) or placebo nightly, and 2 weeks’ placebo run-out. Outcome measures included daily voice recorded sleep diary, Clinical Global Impression of Change (CGIC), WHO-Five Well-being Index (WHO-5), and safety.
Mean nightly sleep duration improved by 43 minutes in the PRM and 16 minutes in the placebo group (mean difference: 27 minutes, 95% CI: −14.4 to 69 minutes; P=0.18; effect size: 0.82) meeting the primary endpoint. Mean sleep latency decreased by 29 minutes with PRM over placebo (P=0.13; effect size: 0.92) and nap duration decreased in the PRM but not placebo group. The variability in sleep onset/offset and latency tended to decrease during PRM but not placebo treatment. The potentially beneficial effects of PRM persisted during the 2 weeks of discontinuation period, consistent with clock stabilizing effects. PRM was well-tolerated, adverse events were of mild or moderate severity and similar between PRM and placebo.
Nightly use of PRM may potentially improve patient-reported sleep difficulties in totally blind individuals trying to adhere to normal social lifestyle. A larger study powered to demonstrate a statistically significant effect is warranted.
PMCID: PMC4319556
biological clock; non-24-hour sleep–wake disorder; sleep; melatonin
24.  Creating Novel Activated Factor XI Inhibitors through Fragment Based Lead Generation and Structure Aided Drug Design 
PLoS ONE  2015;10(1):e0113705.
Activated factor XI (FXIa) inhibitors are anticipated to combine anticoagulant and profibrinolytic effects with a low bleeding risk. This motivated a structure aided fragment based lead generation campaign to create novel FXIa inhibitor leads. A virtual screen, based on docking experiments, was performed to generate a FXIa targeted fragment library for an NMR screen that resulted in the identification of fragments binding in the FXIa S1 binding pocket. The neutral 6-chloro-3,4-dihydro-1H-quinolin-2-one and the weakly basic quinolin-2-amine structures are novel FXIa P1 fragments. The expansion of these fragments towards the FXIa prime side binding sites was aided by solving the X-ray structures of reported FXIa inhibitors that we found to bind in the S1-S1’-S2’ FXIa binding pockets. Combining the X-ray structure information from the identified S1 binding 6-chloro-3,4-dihydro-1H-quinolin-2-one fragment and the S1-S1’-S2’ binding reference compounds enabled structure guided linking and expansion work to achieve one of the most potent and selective FXIa inhibitors reported to date, compound 13, with a FXIa IC50 of 1.0 nM. The hydrophilicity and large polar surface area of the potent S1-S1’-S2’ binding FXIa inhibitors compromised permeability. Initial work to expand the 6-chloro-3,4-dihydro-1H-quinolin-2-one fragment towards the prime side to yield molecules with less hydrophilicity shows promise to afford potent, selective and orally bioavailable compounds.
PMCID: PMC4309560  PMID: 25629509
25.  Ganglioside GD2 expression is maintained upon recurrence in patients with osteosarcoma 
Osteosarcoma is the most common primary malignant bone tumor in children and young adults. Ganglioside GD2 has been previously found on the cell surface in various tumor types, including osteosarcomas.
In this study, forty-nine additional osteosarcoma samples from 14 individual patients were assessed for GD2 expression via immunohistochemistry, of which 47 samples were found to express GD2. In matched samples from patients, GD2 expression seen at initial biopsy was found to persist in 100% of tissues taken at recurrence.
GD2 expression was found to persist upon recurrence. These results suggest a phase 2 trial in children with recurrent osteosarcoma should provide an appropriate read out on the efficacy of anti-GD2 antibody.
Electronic supplementary material
The online version of this article (doi:10.1186/s13569-014-0020-9) contains supplementary material, which is available to authorized users.
PMCID: PMC4311500  PMID: 25642322
Osteosarcoma; Ganglioside GD2; Immunotherapy; Antibody

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