Genetic features of myelodysplastic syndrome and aplastic anemia in pediatric and young adult patients.
The clinical and histopathological distinctions between inherited versus acquired bone marrow failure and myelodysplastic syndromes are challenging. The identification of inherited bone marrow failure/myelodysplastic syndromes is critical to inform appropriate clinical management. To investigate whether a subset of pediatric and young adults undergoing transplant for aplastic anemia or myelodysplastic syndrome have germline mutations in bone marrow failure/myelodysplastic syndrome genes, we performed a targeted genetic screen of samples obtained between 1990-2012 from children and young adults with aplastic anemia or myelodysplastic syndrome transplanted at the Fred Hutchinson Cancer Research Center. Mutations in inherited bone marrow failure/myelodysplastic syndrome genes were found in 5.1% (5/98) of aplastic anemia patients and 13.6% (15/110) of myelodysplastic syndrome patients. While the majority of mutations were constitutional, a RUNX1 mutation present in the peripheral blood at a 51% variant allele fraction was confirmed to be somatically acquired in one myelodysplastic syndrome patient. This highlights the importance of distinguishing germline versus somatic mutations by sequencing DNA from a second tissue or from parents. Pathological mutations were present in DKC1, MPL, and TP53 among the aplastic anemia cohort, and in FANCA, GATA2, MPL, RTEL1, RUNX1, SBDS, TERT, TINF2, and TP53 among the myelodysplastic syndrome cohort. Family history or physical examination failed to reliably predict the presence of germline mutations. This study shows that while any single specific bone marrow failure/myelodysplastic syndrome genetic disorder is rare, screening for these disorders in aggregate identifies a significant subset of patients with inherited bone marrow failure/myelodysplastic syndrome.


Correction of pelvic adduction during total hip arthroplasty reduces variability in radiographic inclination: findings of a randomised controlled trial.
The study aims were to identify the incidence of pelvic adduction during total hip arthroplasty (THA) in lateral decubitus and to determine, when aiming for 35° of apparent operative inclination (AOI), which of 3 operating table positions most accurately obtained a target radiographic inclination (RI) of 42°: (1) horizontal; (2) 7° head-down; (3) patient-specific position based on correction of pelvic adduction. With patients seated on a levelled theatre table, a ruler incorporating a spirit level was used to draw transverse pelvic lines (TPLs) on the skin overlying the pelvis and sacrum. Subsequently, when positioned in lateral decubitus these lines provided a measure of pelvic adduction. 270 participants were recruited, with 90 randomised to each group for operating table position. In all cases target AOI was 35°, aiming to achieve a target RI of 42°. The primary outcome measure was absolute (unsigned) deviation from the target RI of 42°. 266/270 patients demonstrated pelvic adduction (overall mean 4.4°, range 0- 9.2°). No patients demonstrated pelvic abduction. There were significant differences in RI between each of the 3 groups. The horizontal table group displayed the highest mean RI. The patient specific table position group achieved the smallest absolute deviation from target RI of 42°. In lateral decubitus, unrecognised pelvic adduction is common and is an important contributor to unexpectedly high RI. The use of preoperative TPLs helps identify pelvic adduction and its subsequent correction reduces variability in RI. Clinical Trial Protocol number: NCT01831401.


Simple plane wave implementation for photonic crystal calculations.
A simple implementation of plane wave method is presented for modeling photonic crystals with arbitrary shaped 'atoms'. The Fourier transform for a single 'atom' is first calculated either by analytical Fourier transform or numerical FFT, then the shift property is used to obtain the Fourier transform for any arbitrary supercell consisting of a finite number of 'atoms'. To ensure accurate results, generally, two iterating processes including the plane wave iteration and grid resolution iteration must converge. Analysis shows that using analytical Fourier transform when available can improve accuracy and avoid the grid resolution iteration. It converges to the accurate results quickly using a small number of plane waves. Coordinate conversion is used to treat non-orthogonal unit cell with non-regular 'atom' and then is treated by standard numerical FFT. MATLAB source code for the implementation requires about less than 150 statements, and is freely available at http://www.lions.odu.edu/~sguox002.


Usefulness of magnetic resonance imaging with dynamic contrast enhancement and fat suppression in detecting a pancreatic tumor.
The purpose of this study was to compare the value of dynamic magnetic resonance imaging (MRI) and fat suppression in detecting a pancreatic tumor. The subjects were 19 patients with invasive ductal adenocarcinoma and six patients with islet cell tumor where diagnosis was established pathologically. Breath-hold gradient echo images, breath-hold gradient echo images with fat suppression and breath-hold gradient echo images with dynamic enhancement at 1.5 T were obtained for all patients. The efficacies of these three imaging techniques were compared by calculating the contrast-to-noise ratio, as indicative of conspicuousness between a tumor-affected and a normal pancreas. As for adenocarcinoma, our results indicated that the usefulness in detecting the tumor was high, decreasing in the order dynamic contrast images > fat suppression images > plain images, and that the difference between any two of these three types of image was statistically significant. On the other hand, these imaging techniques showed no statistically significant difference in detecting islet cell tumors. In conclusion, dynamic MRI is the best method for detecting pancreatic adenocarcinoma. As the fat suppression technique has the advantage of being non-invasive, this method is suitable for screening studies of pancreatic adenocarcinoma. However, no advantage was recognized in using the fat suppression technique for detecting an islet cell tumor in comparison with plain MRI.


Characterization of ion contents and metabolic responses to salt stress of different Arabidopsis AtHKT1;1 genotypes and their parental strains.
Plants employ several strategies to maintain cellular ion homeostasis under salinity stress, including mediating ion fluxes by transmembrane transport proteins and adjusting osmotic pressure by accumulating osmolytes. The HKT (high-affinity potassium transporter) gene family comprises Na(+) and Na(+)/K(+) transporters in diverse plant species, with HKT1;1 as the only member in Arabidopsis thaliana. Cell-type-specific overexpression of AtHKT1;1 has been shown to prevent shoot Na(+) overaccumulation under salinity stress. Here, we analyzed a broad range of metabolites and elements in shoots and roots of different AtHKT1;1 genotypes and their parental strains before and after salinity stress, revealing a reciprocal relationship of metabolite differences between an AtHKT1;1 knockout line (hkt1;1) and the AtHKT1;1 overexpressing lines (E2586 UAS GAL4 :HKT1;1 and J2731*UAS GAL4 :HKT1;1). Although levels of root sugars were increased after salt stress in both AtHKT1;1 overexpressing lines, E2586 UAS GAL4 :HKT1;1 showed higher accumulation of the osmoprotectants trehalose, gentiobiose, and melibiose, whereas J2731*UAS GAL4 :HKT1;1 showed higher levels of sucrose and raffinose, compared with their parental lines, respectively. In contrast, the knockout line hkt1;1 showed strong increases in the levels of the tricarboxylic acid (TCA) cycle intermediates in the shoots after salt treatment. This coincided with a significant depletion of sugars, suggesting that there is an increased rate of carbon influx into the TCA cycle at a constant rate of C-efflux from the cycle, which might be needed to support plant survival during salt stress. Using correlation analysis, we identified associations between the Na(+) content and several sugars, suggesting that regulation of sugar metabolism is important in plant responses to salinity stress.


The concordance of intraoperative left ventricular wall-motion abnormalities and electrocardiographic S-T segment changes: association with outcome after coronary revascularization. Multicenter Study of Perioperative Ischemia (McSPI) Research Group.
Transesophageal echocardiography (TEE) and Holter electrocardiography (ECG) are used to detect intraoperative ischemia during coronary artery bypass graft surgery (CABG). Concordance of these modalities and sensitivity as indicators of adverse perioperative cardiac outcomes are poorly defined. The authors tried to determine whether routine use of Holter ECG and TEE in patients with CABGs has clinical value in identifying those patients in whom myocardial infarction (MI) is likely to develop. A total of 351 patients with CABG and both ECG- and TEE-evaluable data were examined for the occurrence of ischemia and infarction. The TEE and five-lead Holter ECGs were performed continuously during cardiac surgery. The incidence of MI (creatine kinase-MB > or = 100 ng/ml) within 12 h of arrival in the intensive care [ICU] unit, new ECG Q wave on ICU admission or on the morning of postoperative day 1, or both, were recorded. Electrocardiographic or TEE evidence of intraoperative ischemia was present in 126 (36%) patients. The concordance between modalities was poor (positive concordance = 17%; Kappa statistic = 0.13). Myocardial infarction occurred in 62 (17%) patients, and 32 (52%) of them had previous intraoperative ischemia. Of these, 28 (88%) were identified by TEE, whereas 13 (41%) were identified by ECG. Prediction of MI was greater for TEE compared with ECG. Wall-motion abnormalities detected by TEE are more common than S-T segment changes detected by ECG, and concordance between the two modalities is low. One half of patients with MI had preceding ECG or TEE ischemia. Logistic regression revealed that TEE is twice as predictive as ECG in identifying patients who have MI.


Circulating endothelial progenitor cells are inversely correlated with the median oxygen tension in the tumor tissue of patients with cervical cancer.
Tumor hypoxia leads to adaptive responses in cancer cells, including an induction of vasculogenesis initiated by circulating endothelial progenitor cells (EPCs) and circulating endothelial cells (CECs). The aim of the present study was to correlate the number of EPCs and CECs with the oxygenation of cervical cancer. Blood concentrations of EPCs were detected by FACS analysis with antibodies for CD34 and vascular endothelial growth factor receptor 2 (VEGFR2). CECs were evaluated by double staining for 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine-labeled acetylated low density lipoprotein (Di-LDL) and lectin in a cell culture assay. Ten patients with cervical cancer were compared with ten healthy volunteers. Intratumoral oxygen tension was assessed polarographically with the computerized Eppendorf histography system. Analysis of CEC numbers revealed no difference between patients and controls. However, patients had lower concentrations of CD34-positive hematopoietic stem cells (HSCs) but a significantly higher fraction of EPCs related to the number of HSCs (1.09% versus 0.53%). This fraction was significantly inversely correlated to the median oxygen tension (r = -0.74, p = 0.015). Our study shows for the first time a significant inverse correlation between the fraction of EPCs and intratumoral oxygen tension. We conclude that the fraction of EPCs should be further evaluated as a useful and convenient marker in the prediction of tumor tissue oxygenation.


Avoiding sedentary behaviors requires more cortical resources than avoiding physical activity: An EEG study.
Why do individuals fail to exercise regularly despite knowledge of the risks associated with physical inactivity? Automatic processes regulating exercise behaviors may partly explain this paradox. Yet, these processes have only been investigated with behavioral outcomes (i.e., based on reaction times). Here, using electroencephalography, we investigated the cortical activity underlying automatic approach and avoidance tendencies toward stimuli depicting physical activity and sedentary behaviors in 29 young adults who were physically active or physically inactive but with the intention of becoming physically active. Behavioral results showed faster reactions when approaching physical activity compared to sedentary behaviors and when avoiding sedentary behaviors compared to physical activity. These faster reactions were more pronounced in physically active individuals and were associated with changes during sensory integration (earlier onset latency and larger positive deflection of the stimulus-locked lateralized readiness potentials) but not during motor preparation (no effect on the response-locked lateralized readiness potentials). Faster reactions when avoiding sedentary behaviors compared to physical activity were also associated with higher conflict monitoring (larger early and late N1 event-related potentials) and higher inhibition (larger N2 event-related potentials), irrespective of the usual level of physical activity. These results suggest that additional cortical resources were required to counteract an attraction to sedentary behaviors. Data and Materials [https://doi.org/10.5281/zenodo.1169140]. Preprint [https://doi.org/10.1101/277988].


Optimization and Dose Estimation of Aerosol Delivery to Non-Human Primates.
In pre-clinical animal studies, the uniformity of dosing across subjects and routes of administration is a crucial requirement. In preparation for a study in which aerosolized live-attenuated measles virus vaccine was administered to cynomolgus monkeys (Macaca fascicularis) by inhalation, we assessed the percentage of a nebulized dose inhaled under varying conditions. Drug delivery varies with breathing parameters. Therefore we determined macaque breathing patterns (tidal volume, breathing frequency, and inspiratory to expiratory (I:E) ratio) across a range of 3.3-6.5 kg body weight, using a pediatric pneumotachometer interfaced either with an endotracheal tube or a facemask. Subsequently, these breathing patterns were reproduced using a breathing simulator attached to a filter to collect the inhaled dose. Albuterol was nebulized using a vibrating mesh nebulizer and the percentage inhaled dose was determined by extraction of drug from the filter and subsequent quantification. Tidal volumes ranged from 24 to 46 mL, breathing frequencies from 19 to 31 breaths per minute and I:E ratios from 0.7 to 1.6. A small pediatric resuscitation mask was identified as the best fitting interface between animal and pneumotachometer. The average efficiency of inhaled dose delivery was 32.1% (standard deviation 7.5, range 24%-48%), with variation in tidal volumes as the most important determinant. Studies in non-human primates aimed at comparing aerosol delivery with other routes of administration should take both the inter-subject variation and relatively low efficiency of delivery to these low body weight mammals into account.


Risk factors for specific histopathological types of postmenopausal breast cancer in the NIH-AARP Diet and Health Study.
Risk factor associations for rare breast cancer variants are often imprecise, obscuring differences between tumor types. To clarify differences, we examined risk factors for 5 histological types of breast cancer in the National Institutes of Health-AARP Diet and Health Study. Risk factor information was self-reported. We followed 192,076 postmenopausal women aged 50-71 years from 1995-1996 through 2006. During that time period, 5,334 ductal, 836 lobular, 639 mixed ductal-lobular, 216 mucinous, and 132 tubular breast cancers were diagnosed. Hazard ratios and 95% confidence intervals were estimated using Cox proportional hazards regression. Heterogeneity was evaluated using case-only logistic regression. The strongest differences were for menopausal hormone therapy (Pheterogeneity < 0.01) and age at first birth (Pheterogeneity < 0.01). Risk of tubular cancer in relation to current menopausal hormone therapy (for current use vs. never use, hazard ratio (HR) = 4.39, 95% confidence interval (CI): 2.77, 6.96) was several times stronger than risk of other histological types (range of HRs, 1.39-1.75). Older age at first birth was unassociated with risk of mucinous (for ≥30 years vs. 20-24 years, HR = 0.62, 95% CI: 0.27, 1.42) or tubular (HR = 1.08, 95% CI: 0.51, 2.29) tumors, in contrast to clear positive associations with lobular (HR = 1.82, 95% CI: 1.39, 2.37) and mixed ductal-lobular (HR = 1.87, 95% CI: 1.39, 2.51) tumors. Differing associations for hormonal factors and mucinous and tubular cancers suggest etiologies distinct from those of common breast cancers.


Role of the cytosolic tails of Rift Valley fever virus envelope glycoproteins in viral morphogenesis.
The correct folding, heterodimerization and trafficking of Gn/Gc envelope glycoproteins of Rift Valley fever virus, RVFV (Bunyaviridae and Phlebovirus genus) are essential for Golgi assembly and budding of viral particles. The Gn and Gc carboxy-terminus contain a Golgi targeting and an ER-retrieval signal, respectively. We generated RVFV-like particles with mutations in the cytosolic tails of Gn or Gc and identified regions important for release of infectious particles. The role of specific amino-acids in these regions was further investigated by creating recombinant mutant viruses by reverse-genetics. Residues outside the suspected Golgi targeting motif, i.e. the di-lysine K29-K30 motif and the N43, R44 and I46 residues of the Gn cytosolic domain, appeared important for Golgi localization and RNP packaging. Concerning the Gc tail, replacement of K2 or K3 in the di-lysine motif, had a drastic impact on Gn trafficking and induced an important organelle redistribution and cell remodeling, greatly affecting particle formation and release.


Partition structures, Polya urns, the Ewens sampling formula, and the ages of alleles.
It has recently been shown that the Ewens sampling formula may be generated by a Polya-like urn model. A genealogical proof of this result equates the labelling of balls in the urn to the partition by age of alleles in the sample. This urn construction is shown to be equivalent to the construction of Kingman (Proc. Roy. Soc. London Ser. A 361 (1978), 1-20) using a Poisson-Dirichlet "paintbox" and as a consequence, the partition by ages is seen to be equivalent to the size biased permutation of the Poisson-Dirichlet distribution. This approach unifies and extends many results on ages of alleles, the Polya urn, and the Poisson-Dirichlet distribution. Furthermore the Ewens sampling formula is characterized as being the only partition structure which may be generated by an urn-like mechanism.


Does continuity of care improve patient outcomes?
Continuity of care is a cornerstone of primary care that has been promoted by recent trends in medical education and in the way health care delivery is organized. We sought to determine the effect of sustained continuity of care (SCOC) on the quality of patient care. We conducted a systematic review of all articles in Medline (January 1966 to January 2002), Educational Resources Information Center (ERIC), and PSYCH INFO using the terms "continuity of care" or "continuity of patient care." We identified additional titles of candidate articles by reviewing the bibliographies of articles from our original MEDLINE search, contacting experts in primary care, health care management, and health services research, and by reviewing bibliographies of textbooks of primary care and public health. Two investigators (MDC, SHJ) independently reviewed the full text to exclude articles that did not fulfill search criteria. Articles excluded were those that focused on physicians-in-training, on SCOC in a non-primary care setting, such as an inpatient ward, or on transitions from inpatient to the outpatient setting. We also excluded articles that did not correlate SCOC to a quality of care measure. From 5070 candidate titles, we examined the full text of 260 articles and found 18 (12 cross-sectional studies, 5 cohort studies and 1 randomized controlled trial) that fulfilled our criteria. Five studies focused on patients with chronic illness (eg, asthma, diabetes). No studies documented negative effects of increased SCOC on quality of care. SCOC is associated with patient satisfaction (4 studies), decreased hospitalizations and emergency department visits (7 studies), and improved receipt of preventive services (5 studies). SCOC improves quality of care, and this association is consistently documented for patients with chronic conditions. Programs to promote SCOC may best maximize impact by focusing on populations with chronic conditions.


Low level of Hepatitis B knowledge and awareness among pregnant women in the Kintampo North Municipality: implications for effective disease control.
Over 2 billion people are infected with Hepatitis B virus (HBV) and about 240 million are chronic carriers of the virus. Chronic HBV infection is an important cause of liver cancer. The infectivity of HBV is hundred times higher than the HIV virus yet it receives comparatively little attention in public health. The study assessed knowledge and awareness of HBV among pregnant women in the Kintampo Municipality of Ghana. A cross-sectional survey was conducted among pregnant women attending antenatal clinic in two facilities between September 2010 and November 2010. We performed analysis to determine factors associated with hepatitis B awareness. Forty-one percent of the 504 women were aware of hepatitis B viral infection, 33.5% of the women were able to correctly mention the transmission routes of Hepatitis B. The radio was the most (42%) mentioned source of information on HBV and the least source of information were places of worship (2.7%).After adjusting for other factors, level of education; SSS/SHS and above OR=4.2, P<00.1, 95% CI (2.5, 7.0) and occupation (Civil servant/Student); OR= 3.8, P00.1, 95% CI (1.7, 8.5) were the important predictors of Hepatitis B awareness. There is a low level of knowledge and awareness of HBV among pregnant women in this municipality. This could potentially hamper effective HBV prevention and control in Ghana. Education on hepatitis B need to be included in health promotion activities. The study was funded by KHRC Director's small grant Initiative.


Polybromodiphenyl ethers in mothers and their newborns from a non-occupationally exposed population (Valencia, Spain).
Polybromodiphenyl ethers (PBDEs) were analyzed in blood serum of pregnant women and in cord blood serum of their newborns from a general population cohort (n = 174; Valencia, Spain). The most abundant PBDE congeners identified were BDE 47, BDE 99, BDE 153, BDE 154 and BDE 209. Their cord blood serum concentrations were about 45% of those in maternal serum but after lipid normalization about the same concentrations were observed in both types of samples. Thus, median of total PBDEs was 9.6 ng/g lipid in cord serum (range between not detected and 140 ng/g lipid) and 9.6 ng/g lipid in maternal serum (range between not detected and 120 ng/g lipid). The distributions of these compounds were dominated by BDE 47 in both cases. In cord blood serum the decreasing order of abundance was BDE 47>BDE 99>BDE 209>BDE 153>BDE 154. The congener composition in maternal serum followed a similar trend: BDE 47>BDE 153>BDE 154>BDE 209>BDE 99. The congener concentrations exhibited a higher degree of correlation in cord blood than in maternal serum. Use of the maternal determinants for categorization of the observed maternal and fetal PBDE concentrations only showed significant associations for the levels in umbilical cord. Neonates from rural areas exhibited statistically significantly lower concentrations than those from urban, semi-urban or metropolitan sites. Maternal serum also showed this difference but the higher dispersion of the concentrations in maternal serum did not afford its recognition with statistical significance. The lower qualitative and quantitative variability in the PBDE concentrations of cord blood serum than maternal serum suggest that the latter is reflecting PBDE contributions from a wider diversity of sources than the former whereas cord blood sera seem to represent the long term standing stock of these compounds accumulated in the maternal tissues.


Equity in long-lasting insecticidal nets and indoor residual spraying for malaria prevention in a rural South Central Ethiopia.
While recognizing the recent achievement in the global fight against malaria, the disease remains a challenge to health systems in low-income countries. Beyond widespread consensuses about prioritizing malaria prevention, little is known about the prevailing status of long-lasting insecticidal nets (LLINs) and indoor residual spraying (IRS) across different levels of wealth strata. The aim of this study was to evaluate the socioeconomic related dimension of inequalities in malaria prevention interventions. This study was conducted in July-August 2014 in Adami Tullu district in the South-central Ethiopia, among 6069 households. A cross-sectional data were collected on household characteristics, LLIN ownership and IRS coverage. Principal component analysis technique was used for ranking households based on socioeconomic position. The inequality was measured using concentration indices and concentration curve. Decomposition method was employed in order to quantify the percentage contribution of each socioeconomic related variable on the overall inequality. The proportion of households with at least one LLIN was 11.6 % and IRS coverage was 72.5 %. The Erreygers normalized concentration index was 0.0627 for LLIN and 0.0383 for IRS. Inequality in LLIN ownership was mainly associated with difference in housing situation, household size and access to mass-media and telecommunication service. Coverage of LLIN was low and significant more likely to be owned by the rich households, whereas houses were sprayed equitably. The current mass free distribution of LLINs should be followed by periodic refill based on continuous monitoring data.


Ste12 and Mcm1 regulate cell cycle-dependent transcription of FAR1.
The transcripts of many genes involved in Saccharomyces cerevisiae mating were found to fluctuate during the cell cycle. In the absence of a functional Ste12 transcription factor, both the levels and the cell cycle pattern of expression of these genes were affected. FUS1 and AGA1 levels, which are maximally expressed only in G1-phase cells, were strongly reduced in ste12- cells. The cell cycle transcription pattern for FAR1 was changed in ste12- cells: the gene was still significantly expressed in G2/M, but transcript levels were strongly reduced in G1 phase, resulting in a lack of Far1 protein accumulation. G2/M transcription of FAR1 was dependent on the transcription factor Mcm1, and expression of a gene with Mcm1 fused to a strong transcriptional activation domain resulted in increased levels of FAR1 transcription. The pattern of cell cycle-regulated transcription of FAR1 could involve combinatorial control of Ste12 and Mcm1. Forced G1 expression of FAR1 from the GAL1 promoter resorted the ability to arrest in response to pheromone in ste12-cells. This indicates that transcription of FAR1 in the G1 phase is essential for accumulation of the protein and for pheromone-induced cell cycle arrest.


Preclinical studies of the role of iron oxide magnetic nanoparticles for nonpalpable lesion localization in breast cancer.
One-third of all breast cancers are present as clinically nonpalpable lesions. The current gold standard treatment is surgical excision by wire-guided localization. This technique has patient, technical, and scheduling drawbacks. Alternatives exist but depend on radioisotopes with their legislative and waste management issues. Magnetic nanoparticles (MNPs) have already been successfully used for sentinel lymph node biopsy in breast cancer. We therefore aimed to determine the feasibility of using iron oxide MNPs and a handheld magnetometer for the localization of nonpalpable breast cancers using a preclinical model. We constructed phantom models to assess the relationship between the handheld magnetometer peak readings and the variation in volume of iron oxide MNPs and their depth of injection in a series of porcine and avian tissue models. We also radiolabeled the MNPs with (99m)Tc and alendronate to create the conjugate (99m)Tc-dipicolylamine-alendronate-MNP and used nano-single-photon emission computed tomography-computed tomography to perform imaging to demonstrate localization properties. The handheld magnetometer readings follow a linear relationship with variations in volume of magnetic tracer and a logarithmic relationship with variations in depth of injection of the magnetic tracer at a fixed volume. The application of derived quadratic equations from the R(2) curves of handheld magnetometer signal versus depth allowed calculation of the depth of injection of iron oxide MNPs from the handheld magnetometer readings and demonstrated the predictable behavior of the iron oxide MNPs and the handheld magnetometer. Satisfactory localization characteristics were confirmed in the phantoms and imaged using nano-single-photon emission computed tomography and computed tomography. Iron oxide MNPs demonstrate positive localization characteristics in phantom models with predictable behavior patterns. We suggest that the use of MNPs provides a potential technique for the localization of nonpalpable breast lesions and deserves further exploration in animal and human feasibility studies.


The relationship of neutrophil to lymphocyte ratio with testicular cancer.
To assess the relationship between testicular germ cell tumors (TGCT) and neutrophil to lymphocyte ratio (NLR) and to determine whether this ratio can be used as a serum tumor marker. Sixty-one patients with testicular germ cell tumors were included into the study. Patients were grouped as localized and non-localized. Histologically patients were categorized as seminoma and nonseminomatous germ cell tumors. Complete blood cell count was measured the day before surgery and at the postoperative 1st month. Preoperative and postoperative mean NLR values were compared. Thirty-six patients (59%) had seminomas and 25 patients (41%) had nonseminomatous testicular cancer. Forty-five patients (73.8%) had localized and 16 patients (26.2%) had non-localized testicular cancer. There was a statistically significant difference between preoperative and postoperative mean NLR of the localized patients (p=0.001) but no such difference was detected for non-localized patients (p=0.576). Nineteen patients with localized seminomas had normal preoperative serum tumor markers. There was a significant difference between preoperative and postoperative mean NLR in this group of patients (p=0.010). Twenty-six patients with localized tumors had preoperative increased serum tumor markers which normalized after orchiectomy. Mean NLR of these patients significantly decreased from 3.10 ± 2.13 to 1.62 ± 0.59 postoperatively (p=0.010). NLR appears to be a useful marker for TGCT. It is successful in predicting localized and non-localized disease in early postoperative period.


Towards a Food-Based Intervention to Increase Protein Intakes in Older Adults: Challenges to and Facilitators of Egg Consumption.
Abstract: Background: Dietary protein intake is important for health. Eggs, as a protein-rich food with characteristics that appeal to older adults, may provide opportunities for increasing protein intake. Interventions that focus on the challenges or facilitators that affect a large proportion of the population will be of increased impact on a population-wide scale. This work aimed to investigate the relative importance of a number of challenges to and facilitators of egg consumption in a UK population-wide sample of older adults. A cross-sectional postal questionnaire, measuring habitual egg intake, reasons for eating/not eating eggs and a range of demographic and lifestyle characteristics, was administered by post to 1082 older adults. 230 questionnaires suitable for analysis were returned (110 females, ages 55⁻80+ years). Habitual egg intake ranged from 1⁻89 eggs/month, mean (standard deviation) = 18 (13) eggs/month. Reasons for eating/not eating eggs were reduced using Principal Components Analysis to 23 challenges and facilitators of egg consumption. Regression analyses revealed habitual egg intake to be associated with 10 challenges and facilitators (smallest β = 0.14, p = 0.04), and with protein consumption, age and Body Mass Index (smallest β = 0.14, p = 0.03). Many possibilities for future intervention based on existing challenges or facilitators were found. Our results suggest that strategies to increase egg consumption in older adults should focus on: improving liking, tastiness and adding variety; promoting eggs as an everyday type of food; reducing stereotypes about who does and who does not consume eggs; and promoting eggs for people who have noticed the effects of ageing on their food intake. Strategies that highlight value-for-money may be counterproductive. Future work evaluating the value of these strategies for improving protein intake in this age group would be of value.


Dynamic cyclovergence during vertical translation in humans.
When humans are accelerated along the body vertical, the right and left eyes show oppositely directed torsional modulation (cyclovergence). The origin of this paradoxical response is unknown. We studied cyclovergence during linear sinusoidal vertical motion in healthy humans. A small head-fixed visual target minimized horizontal and vertical motion of the eyes and therefore isolated the torsional component. For stimuli between 1 and 2 Hz (near the natural range of head motion), the phase of cyclovergence with respect to inertial acceleration was 8.7 ± 2.4° (mean ± 95% CI) and the sensitivity (in degrees per second per g) showed a small but statistically significant increase with frequency. These characteristics contrast with those of cycloversion (conjugate torsion) during horizontal (interaural) inertial stimuli at similar frequencies. From these and previous results, we propose that cyclovergence during vertical translation has two sources, one, like cycloversion, from the low-frequency component of linear acceleration, and another, which we term dynamic cyclovergence, with high-pass characteristics. Furthermore, we suggest that this cyclovergence response in humans is a vestige of the response of lateral-eyed animals to vertical linear acceleration of the head.


Effects of phosphatidylserine supplementation on exercising humans.
Phosphatidylserine (PtdSer) is a ubiquitous phospholipid species that is normally located within the inner leaflet of the cell membrane. PtdSer has been implicated in a myriad of membrane-related functions. As a cofactor for a variety of enzymes, PtdSer is thought to be important in cell excitability and communication. PtdSer has also been shown to regulate a variety of neuroendocrine responses that include the release of acetylcholine, dopamine and noradrenaline. Additionally, PtdSer has been extensively demonstrated to influence tissue responses to inflammation. Finally, PtdSer has the potential to act as an effective antioxidant, especially in response to iron-mediated oxidation. The majority of the available research that has investigated the effects of PtdSer supplementation on humans has concentrated on memory and cognitive function; patients experiencing some degree of cognitive decline have traditionally been the main focus of investigation. Although investigators have administered PtdSer through intravenous and oral routes, oral supplementation has wider appeal. Indeed, PtdSer is commercially available as an oral supplement intended to improve cognitive function, with recommended doses usually ranging from 100 to 500 mg/day. The main sources that have been used to derive PtdSer for supplements are bovine-cortex (BC-PtdSer) and soy (S-PtdSer); however, due to the possibility of transferring infection through the consumption of prion contaminated brain, S-PtdSer is the preferred supplement for use in humans. Although the pharmacokinetics of PtdSer have not been fully elucidated, it is likely that oral supplementation leads to small but quantifiable increases in the PtdSer content within the cell membrane.A small number of peer-reviewed full articles exist that investigate the effects of PtdSer supplementation in the exercising human. Early research indicated that oral supplementation with BC-PtdSer 800 mg/day moderated exercise-induced changes to the hypothalamo-pituitary-adrenal axis in untrained participants. Subsequently, this finding was extended to suggest that S-PtdSer 800 mg/day reduced the cortisol response to overtraining during weight training while improving feeling of well-being and decreasing perceived muscle soreness. However, equivocal findings from our laboratory might suggest that the dose required to undertake this neuroendocrine action may vary between participants.Interestingly, recent findings demonstrating that short-term supplementation with S-PtdSer 750 mg/day improved exercise capacity during high-intensity cycling and tended to increase performance during intermittent running might suggest an innovative application for this supplement. With the findings from the existing body of literature in mind, this article focuses on the potential effects of PtdSer supplementation in humans during and following exercise.


Getting used to it: the adaptive global utility model.
This paper expands the standard model of utility maximization to endogenize the ubiquitous phenomenon of adaptation. We assume that total utility is an aggregate function of the utility associated with different domains of life, with relative weights that are optimized according to the effort that the individual expends on producing utility in each domain. Comparative statics from the general maximization problem demonstrate that the traditional Slutsky equation should incorporate an additional response term to account for adaptation processes. Our adaptive global utility maximization model can be used to explain responses to changes in health.


Circadian regulation of depression: A role for serotonin.
Synchronizing circadian (24 h) rhythms in physiology and behavior with the environmental light-dark cycle is critical for maintaining optimal health. Dysregulation of the circadian system increases susceptibility to numerous pathological conditions including major depressive disorder. Stress is a common etiological factor in the development of depression and the circadian system is highly interconnected to stress-sensitive neurotransmitter systems such as the serotonin (5-hydroxytryptamine, 5-HT) system. Thus, here we propose that stress-induced perturbation of the 5-HT system disrupts circadian processes and increases susceptibility to depression. In this review, we first provide an overview of the basic components of the circadian system. Next, we discuss evidence that circadian dysfunction is associated with changes in mood in humans and rodent models. Finally, we provide evidence that 5-HT is a critical factor linking dysregulation of the circadian system and mood. Determining how these two systems interact may provide novel therapeutic targets for depression.


Antipredator vigilance of juvenile and adult thirteen-lined ground squirrels and the role of nutritional need.
Juvenile thirteen-lined ground squirrels, Spermophilus tridecemlineatus, are less vigilant (i.e. they spend less time visually scanning the environment) than adults. To determine whether nutritional need was a potential cause of this difference, we supplemented two groups of free-ranging juveniles during the predispersal stage, while juveniles were still near and around the natal burrows. The high-energy food group (HEF: 11 squirrels) received peanut butter and oats while the low-energy food group (LEF: seven squirrels) received lettuce. Adults (14 squirrels) were also supplemented, but due to their greater home range sizes, it was not feasible to classify them as either HEF or LEF. To evaluate the effect of supplementation on antipredator vigilance, the behavioural act of visually scanning for predators, we videotaped individuals while they were foraging above ground during 5-min observation periods. Each squirrel was observed and weighed during three time periods over 23 days. From the videotape, we extracted measures of time spent vigilant, locomoting and foraging. All three categories of squirrels gained mass over the study period, but the HEF juveniles rapidly exceeded that of the LEF juveniles. Early in the study, LEF and HEF juveniles did not significantly differ in either body mass or time budgets, and, initially, both juvenile groups were similar to adults in the amount of time devoted to vigilance. Later in the study, the behaviour of HEF juveniles closely resembled that of adults (increased time devoted to vigilance and decreased time devoted to foraging), while LEF juveniles decreased vigilance and increased their foraging time. This study indicates that for thirteen-lined ground squirrels the lower vigilance of juveniles is due, at least in part, to the greater nutritional needs of young animals with consequent increases in foraging, which is largely incompatible with vigilance. Copyright 2000 The Association for the Study of Animal Behaviour.


Qualitative plasma PCR assay (AMPLICOR CMV test) versus pp65 antigenemia assay for monitoring cytomegalovirus viremia and guiding preemptive ganciclovir therapy in allogeneic stem cell transplantation.
The performances of a commercially available qualitative plasma PCR assay (AMPLICOR CMV test; Roche Diagnostics) and the pp65 antigenemia assay (AG) were evaluated for the monitoring of cytomegalovirus (CMV) viremia in 43 allogeneic stem cell transplant recipients. In addition, the suitabilities of both assays for triggering the initiation of preemptive ganciclovir therapy were assessed. A total of 37 CMV viremic episodes were detected in 28 patients. Positivity of plasma PCR testing in one or more consecutive specimens was the only marker of CMV viremia in 18 of the 37 episodes (PCR positive and AG negative, n = 50 specimens). Five episodes were diagnosed on the basis of a single positive AG result (AG positive and PCR negative, n = 5 specimens); both assays were eventually positive (PCR positive and AG positive, n = 27 specimens) for 14 viremic episodes; for these episodes, conversion of the PCR assay result to a positive result occurred an average of 1 week before conversion of the AG result. Overall, the concordance between the two methods was 90%, and the sensitivities of the plasma PCR assay and AG for the detection of CMV viremic episodes were 86.5 and 51.3%, respectively. Two patients who tested positive by both assays simultaneously progressed to CMV end-stage organ disease, despite the initiation of preemptive ganciclovir therapy. Conversion of the AG result to a negative result upon administration of preemptive ganciclovir therapy occurred a median of 7.5 days earlier than conversion of the plasma PCR assay result. Nineteen of the 28 patients with CMV viremia received AG-guided preemptive ganciclovir therapy; had the positivity of the plasma PCR assay triggered the initiation of preemptive therapy, 9 additional patients would have been unnecessarily treated since none of them developed CMV end-stage organ disease. Although the AMPLICOR CMV assay is more sensitive than AG, the latter appears to be more suitable both for guiding the initiation of preemptive therapy and for monitoring a patient's response to antiviral therapy.


Cancer-Related Pain Management and the Optimal Use of Opioids.
Pain relief is vital to the treatment of cancer. Despite the widespread use and recognition of clinical recommendations for the management of cancer-related pain, avoidable suffering is still prevalent in patients with malignant disease. A gap exists between what is known about pain medical management and actual practices of patients, caregivers, healthcare professionals and institutions. Opioids are the pillar of the medical management of moderate to severe pain. The prescription of opioid analgesics - by a registered medical practitioner for absolute pain control - is a legitimate practice. In this article we look at patients' fears and physicians' general hesitations towards morphine and alike. We examine misconceptions that yield fallacies on the therapeutically use of opioids and, therefore, sustain inadequate pain management.


A Novel Comprehensive Clinical Stratification Model to Refine Prognosis of Glioblastoma Patients Undergoing Surgical Resection.
Despite recent discoveries in genetics and molecular fields, glioblastoma (GBM) prognosis still remains unfavorable with less than 10% of patients alive 5 years after diagnosis. Numerous studies have focused on the research of biological biomarkers to stratify GBM patients. We addressed this issue in our study by using clinical/molecular and image data, which is generally available to Neurosurgical Departments in order to create a prognostic score that can be useful to stratify GBM patients undergoing surgical resection. By using the random forest approach [CART analysis (classification and regression tree)] on Survival time data of 465 cases, we developed a new prediction score resulting in 10 groups based on extent of resection (EOR), age, tumor volumetric features, intraoperative protocols and tumor molecular classes. The resulting tree was trimmed according to similarities in the relative hazard ratios amongst groups, giving rise to a 5-group classification tree. These 5 groups were different in terms of overall survival (OS) (p < 0.000). The score performance in predicting death was defined by a Harrell's c-index of 0.79 (95% confidence interval [0.76-0.81]). The proposed score could be useful in a clinical setting to refine the prognosis of GBM patients after surgery and prior to postoperative treatment.


Epidemiology of hepatitis C infection and its public health implications in Puerto Rico.
Hepatitis C infection is the most common chronic blood-borne pathogen in the United States associated with liver cirrhosis and hepatocellular carcinoma and is the leading reason for liver transplantation. It has been estimated that hepatitis C infection may lead to a substantial health and economic burden over the next 10 to 20 years. The prevalence of hepatitis C virus (HCV) infection varies worldwide, with an estimated overall prevalence of 3%. However, the only available data of hepatitis C in the general population of Puerto Rico suggest an elevated prevalence of hepatitis C infection in the municipality of San Juan (6.3%) in comparison with estimates for the adult population residing in the United States (0.9%-3.9%). Much of the inter-region variability in the prevalence of hepatitis C can be attributable to the frequency and extent to which different risk factors have contributed to the transmission of the virus. Established risk factors for infection include injection drug use, transfusion of blood and solid organ transplantation from infected donors prior to July 1992 and blood clotting products before 1987, occupational injury, vertical transmission, sex with an HCV infected partner, and multiple sexual partners. Other potential exposures for infection that have been investigated in epidemiologic studies include history of intranasal cocaine use, sharing of contaminated equipment and personal care items, tattooing, body piercing, imprisonment, acupuncture, and use of contaminated healthcare instruments. The high incidence of AIDS in Puerto Rico and the large prevalence observed in Puerto Rican inmates and in adults residing in the municipality of San Juan indicate that HCV infection is an emerging public health concern. From a public health perspective, potential targets for intervention to decrease the spread of HCV infection, ongoing surveillance, increased clinician awareness of disease reporting systems and the epidemiology and management of hepatitis C, availability of diagnosis and treatment facilities, and recognition of the need for local resources will be of paramount importance to face this silent infection in Puerto Rico.


Reproducibility and variance of a stimulation-induced hemodynamic response in barrel cortex of awake behaving mice.
The present work evaluated the reproducibility and variance of the cerebral blood flow (CBF) response to natural whisker stimulation in the barrel cortex of awake behaving mice. The animal was placed on an air float ball that allowed the animal to walk, while the head of the animal was fixed in a custom-made stereotactic apparatus. Dynamic CBF changes in the barrel cortex and animal locomotion were simultaneously monitored with laser-Doppler flowmetry (LDF) and an optical motion sensor that detected the rotation distance of the ball, respectively. Whisker stimulation-induced CBF measured under daytime and nighttime conditions showed consistent responses (24% and 23% of the pre-stimulus baseline, respectively), whereas the amount of locomotion was 1.4 times higher during nighttime relative to daytime. Repeated longitudinal experiments over 7 days showed a reproducible, evoked CBF (13-26% relative to the baseline among 7 animals). The mean of the variance coefficient (i.e., standard deviation divided by mean) across multiple days was 0.11 and 0.75 for evoked CBF and locomotion, respectively. These results showed reproducible and reliable measurements of longitudinal CBF response in behaving mice regardless of day-to-day variations in locomotion. Furthermore, we confirmed that the CBF response to whisker stimulation was well localized and reproducible, measured with laser speckle imaging under awake condition. The results further show the capability of long-term hemodynamic imaging in normal and disease-model mice, which is of particular importance for understanding the longitudinal changes and plasticity of neurovascular coupling and behavioral performances such as during growth, development and aging.


Pathological changes induced by repeated percutaneous transluminal coronary angioplasty.
The histopathological appearances of seven coronary arteries obtained from four patients after repeated percutaneous transluminal coronary angioplasty were analysed. A complex picture was found; typically there were ruptured atherosclerotic plaques, plaque dissection, and a fibrous tissue response. The histopathological appearance of older and more recent fibrous lesions was different. Older lesions contained more collagen and elastin fibres, whereas recent ones had more loosely arranged connective tissue containing abundant glycosaminoglycan and readily identifiable cells. The fibrous tissues tended to be damaged at the sites of previous injury and where the vessel wall was thinnest. In five of the seven arteries there was evidence of a repeated fibrous response to injury with partial or total rupture of the original media. In one instance a repair response within a pre-existing atherosclerotic plaque had caused restenosis. The results indicate that restenosis after repeated percutaneous transluminal coronary angioplasty, like restenosis after a first procedure, is mainly the result of fibrocellular tissue response to injury of the wall tissues. Because older (that is more mature) repair tissue contains fewer cells and more connective elements than younger repair tissue (that is the loosely arranged connective tissue found soon after angioplasty), when it is disrupted by a further angioplasty procedure it is less capable of producing tissue that will obstruct the lumen. This may explain why in the majority of patients with restenosis repeated percutaneous transluminal coronary angioplasty is successful. The present study also showed that occasionally plaque haemorrhages may become organised and incorporated into the pre-existing atherosclerotic lesion.


Integrated Platform for Monitoring Single-cell MAPK Kinetics in Computer-controlled Temporal Stimulations.
Extracellular response kinase (ERK) is one of the key regulator of cell fate, such as proliferation, differentiation and cell migration. Here, we propose a novel experimental pipeline to learn ERK kinetics by temporal growth factor (GF) stimulation. High signal-to-noise ratio of genetically encoded Fluorescence resonance energy transfer (FRET) biosensor enables to get a large number of single-cell ERK activity at each time point, while computer-controlled microfluidics fine-tune the temporal stimulation. Using this platform, we observed that static Epidermal growth factor (EGF) stimulation led to transient ERK activation with a significant cell-to-cell variation, while dynamic stimulation of 3' EGF pulse led to faster adaptation kinetics with no discrepancy. Multiple EGF pulses retriggered ERK activity with respect to frequency of stimulation. We also observed oscillation of ERK activity of each cell at basal state. Introducing of Mitogen-activated protein kinase kinase (MEK) inhibitor, U0126, was not only dropping the average of basal activity for 7.5%, but also diminishing oscillatory behavior. Activity level raised up when inhibitor was removed, followed by transient peak of ERK kinetics. We expect this platform to probe Mitogen-associated protein kinase (MAPK) signaling network for systems biology research at single cellular level.


Assessing the onset of antidepressant-induced sexual dysfunction using interactive voice response technology.
Sexual dysfunction is a symptom of major depression, as well as a common complication of treatment with many classes of antidepressants. Nonetheless, the various forms of sexual dysfunction continue to be underreported in clinical practice, despite the availability of validated scales such as the Changes in Sexual Functioning Questionnaire (CSFQ). The current study was designed to evaluate the validity of obtaining CSFQ data using interactive voice response (IVR) technology. Sexually active, healthy male volunteers (N = 99; mean age of 31 years) were randomly assigned to 3 weeks of double-blind, parallel-group treatment with paroxetine (20 mg/day); CP-448,187 (3 mg/day); or placebo. Patients completed both paper-and-pencil and IVR versions of the 14-item CSFQ at baseline and on treatment days 8, 15, and 21. Additional IVR assessments were obtained at days 2, 4, and 6, permitting assessment of changes between office visits. This study was conducted between March and May 2001. The overall correlation between the paper and IVR CSFQ total score was r = 0.96 (p < .0001). Similarly, high correlations were found between paper and IVR assessment methods on the individual CSFQ subscales: pleasure (r = 0.88), frequency (r = 0.88), interest (r = 0.93), arousal (r = 0.89), and orgasm (r = 0.92; p < .0001 for all comparisons). Both assessment methods were able to detect a statistically significant between-group difference in sexual functioning by day 8, which remained significant throughout the remainder of the study. Both assessment methods found SSRI-related sexual dysfunction to include significant effects on all CSFQ domains. Assessments using IVR collected from subjects at home on days 2, 4, and 6 identified onset of sexual dysfunction by day 4, before it was detected during scheduled office visits. Interactive voice response assessment of sexual dysfunction on the CSFQ was found to be highly correlated with previously validated paper-and-pencil assessment. Interactive voice response provides a valid, easy-to-administer alternative method for obtaining systematic data on the impact of antidepressant treatment on sexual functioning. More frequent assessment by IVR enables more precise evaluation of symptom onset.


The association between baroreflex sensitivity and blood pressure in children.
Low baroreflex sensitivity (BRS) and heart rate variability (HRV) are associated with the pathogenesis of adult hypertension. However, limited information exists about the negative consequences of elevated childhood blood pressure (BP) and autonomic regulation. Additionally, there are developmental changes in autonomic regulation throughout puberty, yet studies have not appropriately accounted for this. The objective of this study was to investigate BRS and HRV in children with 2 different BP profiles, while controlling for the effects of maturation, age, sex, and body composition. A sample of 11- to 14-year-old participants were divided into 2 BP groups: high BP (HBP; ≥95th percentile; n = 21) and normal BP (NBP; <90th percentile; n = 85). Automated BP was measured at 2 time points. In lab-based testing, 5 min of beat-to-beat BP (Finapres) and R-R interval (RRI) were recorded (standard electrocardiogram) after 15 min of supine rest. Spectral indices were computed using fast Fourier transform, and transfer function analysis was used to compute BRS. High frequency (HF) and low frequency (LF) power spectral areas were set to 0.15-0.4 Hz and 0.04-0.15 Hz, respectively, and BRS was determined for the LF area. After adjustment for age, sex, maturation, and body composition, BRS (p = 0.04), LF (p = 0.008), and HF (p = 0.01) RRI variability, and RRI total power (p = 0.005) were lower in the HBP than in the NBP participants. As well, the LF/HF systolic BP variability ratio was higher in the HBP than in the NBP group (p = 0.03). Despite their young age, these children with high, yet not clinically hypertensive BP, display reduced autonomic regulation.


Scaling up pediatric HIV care and treatment in Africa: clinical site characteristics associated with favorable service utilization.
To improve pediatric enrollment and retention in HIV treatment programs in Africa, we examined factors associated with service utilization within the Elizabeth Glaser Pediatric AIDS Foundation program in Côte d'Ivoire, Mozambique, South Africa, Tanzania and Zambia. We retrospectively reviewed characteristics of clinical sites providing HIV treatment services within our program. For each site, favorable pediatric program outcomes were defined as a cumulative number or percentage of pediatric enrollment in care or antiretroviral therapy (ART) more than the pooled median value or an attrition rate less than 10%. We compared proportions of sites with favorable outcomes among those with or without selected characteristics. Adjusted odds ratios (aORs) and 95% confidence intervals (CIs) were determined using logistic regression analyses, accounting for potential confounding factors. Over 4 years, 33,331 children were enrolled, including 18,255 on ART, across 220 sites. Characteristics associated with favorable pediatric enrollment were nutritional support (aOR = 8.9; CI: 2.8 to 28.4), linkages with associations of people living with HIV (aOR = 4.2; CI: 1.8 to 9.5), early infant diagnosis (aOR = 3.3; CI: 1.5 to 7.1), and on-site prevention of mother-to-child transmission services (aOR = 3.1; CI: 1.0 to 11.1). Similarly, linkages with people living with HIV, early infant diagnosis, and prevention of mother-to-child transmission were associated with high proportion of children on ART younger than 2 years of age. Home-based care was associated with low pediatric attrition rates (aOR = 2.9; CI: 1.4 to 5.8). Certain site characteristics were associated with favorable pediatric enrollment and retention in our program. Expanding these characteristics to improve pediatric HIV treatment in Africa warrants further evaluation.


[Estimating the frequency of induced abortion: a comparison of two methods].
To compare two methods-the "ballot box" method and the "indirect questioning" method-for estimating the frequency of induced abortions in population-based studies. A cross-sectional population-based study was conducted with a representative sample of 3 002 women between 15 and 49 years of age living in the city of Pelotas, Rio Grande do Sul, Brazil. The women were selected through multistage sampling and randomly assigned to answer questions concerning induced abortion with one of the two methods, after they had answered a general questionnaire that collected socioeconomic and demographic information. With the "ballot box" method, women received a small piece of paper containing direct questions on abortion. Each woman marked her answers on the paper and then deposited it into a small "ballot box" carried by the interviewer, thus assuring the confidentiality of the responses. With the second method, the interviewer verbally asked the woman a series of questions that indirectly inquired about abortion. Among the women assigned to the ballot box method, 7.2% reported having induced at least one abortion, versus 3.8% of the women assigned to the indirect questioning method. The ratio between the two methods was 1.89 (95% confidence interval: 1.39 to 2.60; P < 0.001). Of the women surveyed who were 45-49 years old (at the end of their reproductive life) and answered using the ballot box method, 12% of them reported having had at least one induced abortion in their lifetime. Among the women who answered using the ballot box method, the main reasons they gave for having had an abortion were economic difficulties, being too young, and being single. Approximately half of the women responding to the ballot box method questions reported they had used unsafe procedures to induce abortion, with 13% of them using misoprostol. The ballot box method was suitable for studying the frequency of induced abortion and for researching other topics that may lead to underreporting by the persons interviewed, especially in population-based samples. Rephrasing the ballot box method question about abortion (for example, eliminating the word "child") would probably increase the method's sensitivity, that is, its ability to detect induced abortions that had occurred.


4-Alkylated Silver-N-Heterocyclic Carbene (NHC) Complexes with Cytotoxic Effects in Leukemia Cells.
Computational chemistry has shown that backbone-alkylated imidazoles ought to be efficient ligands for transition metal catalysts with improved carbene-to-metal donation. In this work, such alkylated imidazoles were synthesized and complexed with silver(I) by means of an eight/nine-step synthetic pathway we devised to access a new class of biologically active silver complexes. The synthesis involves selective iodination of the imidazole backbone, followed by Sonogashira coupling to replace the backbone iodine. The installed alkyne moiety is then subjected to reductive hydrogenation with Pearlman's catalyst. The imidazole N1 atom is arylated by the palladium-catalyzed Buchwald N-arylation method. The imidazole N3 position was then methylated with methyl iodine, whereupon the synthesis was terminated by complexation of the imidazolium salt with silver(I) oxide. The synthetic pathway provided an overall yield of ≈20 %. The resulting complexes were tested in vitro against HL60 and MOLM-13 leukemic cells, two human-derived cell lines that model acute myeloid leukemia. The most active compounds exhibiting low IC50 values of 14 and 27 μM, against HL60 and MOLM-13 cells, respectively. The imidazole side chain was found to be essential for high cytotoxicity, as the imidazole complex bearing a C7 side chain at the 4-position was four- to sixfold more potent than the corresponding imidazole elaborated with a methyl group.


Comparing microleakage and layering methods of silorane-based resin composite in class V cavities using confocal microscopy: An in vitro study.
This in vitro study compared the effects of different layering techniques on the microleakage of silorane-based resin composite using confocal laser scanning microscope. Forty caries free premolars extracted for orthodontic reasons were used. A class V cavity was prepared on the buccal surface in each of the premolars, with the gingival margin of the cavity being 1 mm above cementoenamel junction. The cavities were restored with a silorane-based resin composite (Filtek(™) P90 Silorane Low Shrink Restorative, 3M ESPE) using two different layering techniques - split incremental and oblique layering technique. All samples were subjected to 1000 thermal cycles of 5°C/55°C in water with a 30 second dwell time, and after the procedure, the teeth were immersed in 0.6% aqueous rhodamine dye for 48 hours. Sectioned samples were examined under a Confocal Fluorescence Imaging Microscope (Leica TCS-SP5, DM6000-CFS) at 10× magnification, and microleakage scores were analyzed statistically using paired "t" test and Mann-Whitney test. Width of interface between the tooth surface and resin composite was measured using a digital scale (Snagit digital scale). Microleakage was seen along the entire perimeter of restoration irrespective of the layering technique used. The microleakage score was same in both the groups. Statistical analysis of width of interface showed significant difference between the two layering techniques. The width was significantly less in split incremental technique, indicating less polymerization shrinkage. This in vitro study showed that the silorane-based resin composite shows microleakage irrespective of the layering technique used for class V cavities. However, this problem can be minimized significantly by using split incremental technique for restoration of class V lesions.


Comparative sequence analysis of MONOCULM1-orthologous regions in 14 Oryza genomes.
Comparative genomics is a powerful tool to decipher gene and genome evolution. Placing multiple genome comparisons in a phylogenetic context improves the sensitivity of evolutionary inferences. In the genus Oryza, this comparative approach can be used to investigate gene function, genome evolution, domestication, polyploidy, and ecological adaptation. A large genomic region surrounding the MONOCULM1 (MOC1) locus was chosen for study in 14 Oryza species, including 10 diploids and 4 allotetraploids. Sequencing and annotation of 18 bacterial artificial chromosome clones for these species revealed highly conserved gene colinearity and structure in the MOC1 region. Since the Oryza radiation about 14 Mya, differences in transposon amplification appear to be responsible for the different current sizes of the Oryza genomes. In the MOC1 region, transposons were only conserved between genomes of the same type (e.g., AA or BB). In addition to the conserved gene content, several apparent genes have been generated de novo or uniquely retained in the AA lineage. Two different 3-gene segments have been inserted into the MOC1 region of O. coarctata (KK) or O. sativa by unknown mechanism(s). Large and apparently noncoding sequences flanking the MOC1 gene were observed to be under strong purifying selection. The allotetraploids Oryza alta and Oryza minuta were found to be products of recent polyploidization, less than 1.6 and 0.4 Mya, respectively. In allotetraploids, pseudogenization of duplicated genes was common, caused by large deletions, small frame-shifting insertions/deletions, or nonsense mutations.


Polymer stabilization of cholesteric liquid crystals in the oblique helicoidal state.
Electrical control of the pitch has been reported in a variant of the cholesteric liquid crystal phase composed of chiral dopants and liquid crystal dimers with a bent conformation, such as CB7CB. For a finite range of applied electric field, the dimeric mesogens assume an oblique helicoidal structure, in which the helical axis is aligned along the electric field and the local director is tilted towards the helical axis (rather than being perpendicular to it). An electric field can directly regulate the periodicity (pitch), allowing reconfiguration of the optical response from a scattering or transparent state to a reflective state. Here, we employ po stabilization to retain the oblique helicoidal state absent an applied field. The polymer stabilized oblique helicoidal structures were investigated under various conditions and material compositions. With polymer stabilization, the magnitude of the selective reflection is found to be dependent on the strength of the applied field. Comparison of the electro-optical response of samples with and without a polymer network elucidates the relative role of boundary conditions, anchoring strength, and elastic energy on the stability of the oblique helicoidal state.


Immunological quantitation of thymidylate synthase using the monoclonal antibody TS 106 in 5-fluorouracil-sensitive and -resistant human cancer cell lines.
Thymidylate synthase (TS) (EC 220.127.116.11) is an important cellular target for the fluoropyrimidine cytotoxic drugs that are widely used in the treatment of solid tumors. Using the TS 106 monoclonal antibody to human TS, we have compared the immunological quantitation of TS by Western immunoblot and immunofluorescent techniques to the conventional biochemical 5-fluorodeoxyuridine monophosphate binding assay in a panel of 5-fluorouracil (5-FU)-sensitive and -resistant human cancer cell lines. Densitometric quantitation of TS 106-labeled Western immunoblot analysis of cell lysates from two 5-FU-resistant colon carcinoma cell lines, NCI H630R1 and NCI H630R10, revealed 12.8- and 16-fold increases in TS, respectively, compared to the parent 5-FU-sensitive NCI H630 colon cell line. By biochemical analysis, the TS level was 15- and 23-fold higher, respectively, in these resistant cell lines. Similarly, immunoblot analysis of cell lysates from two 5-FU-resistant breast cancer cell lines, MCF-Ad5 and MCF-Ad10, detected a 2.3- and 6.3-fold increase in TS, respectively, compared to the parent MCF-7 cell line. By biochemical assay the TS activity was 1.8- and 7.0-fold higher in these resistant breast cell lines. Western immunoblotting analysis revealed a 35-fold range of TS protein concentrations among the 10 cell lines examined, compared to a 38-fold range demonstrated by the biochemical assay. Direct comparison of Western blotting and the biochemical assay revealed a highly significant correlation (r2 = 0.93) between the two assays. Moreover, using the monoclonal antibody TS 106, the Western blotting technique was capable of detecting TS protein levels as low as 0.3 fmol in cellular lysates. Quantitation of TS in intact cells by immunofluorescent TS labeling and flow cytometric analysis was performed using three of the cell lines, NCI H630, NCI H630R10, and MCF-Ad10. This revealed a 26-fold increase in TS in the 5-FU-resistant NCI H630R10 line compared to the parent NCI H630 line and a 3.5-fold increase in TS compared to the 5-FU-resistant MCF-Ad10 breast cell line. The 5-FU-resistant MCF-Ad10 breast cell line, in turn, displayed a 7.7-fold increase in TS, compared to the 5-FU-sensitive NCI H630 cell lines. TS immunofluorescent analysis was capable of measuring TS within individual cells. The development of these immunological assays using an anti-TS monoclonal antibody will facilitate the quantitation of TS in cell lines and tissue samples.


Haploinsufficiency for DNA methyltransferase 3A predisposes hematopoietic cells to myeloid malignancies.
The gene that encodes de novo DNA methyltransferase 3A (DNMT3A) is frequently mutated in acute myeloid leukemia genomes. Point mutations at position R882 have been shown to cause a dominant negative loss of DNMT3A methylation activity, but 15% of DNMT3A mutations are predicted to produce truncated proteins that could either have dominant negative activities or cause loss of function and haploinsufficiency. Here, we demonstrate that 3 of these mutants produce truncated, inactive proteins that do not dimerize with WT DNMT3A, strongly supporting the haploinsufficiency hypothesis. We therefore evaluated hematopoiesis in mice heterozygous for a constitutive null Dnmt3a mutation. With no other manipulations, Dnmt3a+/- mice developed myeloid skewing over time, and their hematopoietic stem/progenitor cells exhibited a long-term competitive transplantation advantage. Dnmt3a+/- mice also spontaneously developed transplantable myeloid malignancies after a long latent period, and 3 of 12 tumors tested had cooperating mutations in the Ras/MAPK pathway. The residual Dnmt3a allele was neither mutated nor downregulated in these tumors. The bone marrow cells of Dnmt3a+/- mice had a subtle but statistically significant DNA hypomethylation phenotype that was not associated with gene dysregulation. These data demonstrate that haploinsufficiency for Dnmt3a alters hematopoiesis and predisposes mice (and probably humans) to myeloid malignancies by a mechanism that is not yet clear.


Advances in Pediatric Surgical Education: A Critical Appraisal of Two Consecutive Minimally Invasive Pediatric Surgery Training Courses.
Mandates for improved patient safety and increasing work hour restrictions have resulted in changes in surgical education. Educational courses increasingly must meet those needs. We sought to determine the experience, skill level, and the impact of simulation-based education (SBE) on two cohorts of pediatric surgery trainees. After Institutional Review Board (IRB) exempt determination, a retrospective review was performed of evaluations for an annual advanced minimally invasive surgery (MIS) course over 2 consecutive years. The courses included didactic content and hands-on skills training. Simulation included neonatal/infant models for rigid bronchoscopy-airway foreign body retrieval, laparoscopic common bile duct exploration, and real tissue diaphragmatic hernia (DH), duodenal atresia (DA), pulmonary lobectomy, and tracheoesophageal fistula models. Categorical data were analyzed with chi-squared analyses with t-tests for continuous data. Participants had limited prior advanced neonatal MIS experience, with 1.95 ± 2.84 and 1.16 ± 1.54 prior cases in the 2014 and 2015 cohorts, respectively. The 2015 cohort had significantly less previous experience in lobectomy (P = .04) and overall advanced MIS (P = .007). Before both courses, a significant percentage of participants were not comfortable with DH repair (39%-42%), DA repair (50%-74%), lobectomy (34%-43%), and tracheoesophageal fistula repair (54%-81%). After course completion, > 60% of participants reported improvement in comfort with procedures and over 90% reported that the course significantly improved their perceived ability to perform each operation safely. Pediatric surgery trainees continue to have limited exposure to advanced MIS during clinical training. SBE results in significant improvement in both cognitive knowledge and trainee comfort with safe operative techniques for advanced MIS.


Hereditary medullary thyroid cancer: age-appropriate thyroidectomy improves disease-free survival.
Twenty-five percent of medullary thyroid cancer (MTC) cases are hereditary. The ideal age for prophylactic thyroidectomy is based on the specific RET mutation involved. The purpose of this study was to determine whether such age-appropriate prophylactic thyroidectomy results in improved disease-free survival. Twenty-eight patients underwent thyroidectomy for hereditary MTC at our institution. Age-appropriate thyroidectomy was defined according to the North American Neuroendocrine Tumor Society (NANETS) guidelines. Patients who had age-appropriate surgery (group 1, n = 9) were compared to those who had thyroidectomy past the recommended age (group 2, n = 19). The mean age was 13 ± 2 years, and 61 % were female. Patients in group 1 were younger than in group 2 (4 ± 1 vs. 17 ± 2 years, p < 0.01). There were no significant differences in gender or RET mutation types between these two groups. Group 1 patients were cured with no disease recurrence compared with group 2 patients who had a 42 % recurrence rate (p = 0.05). Subanalysis of group 2 identified that patients who underwent surgery without evidence of disease did so at a shorter period following the guidelines compared with those who underwent therapeutic surgery (2 ± 2 vs. 16 ± 2 years, p = 0.01) and had longer disease-free survival (100 vs. 27 %, p = 0.005). Patients with hereditary MTC should undergo age-appropriate thyroidectomy based on RET mutational status to avoid recurrence. Patients who are past the recommended age should have surgery as early as possible to improve disease-free survival.


A multi-level analysis of individual- and school-level correlates of physical fitness in children.
Children's physical fitness is potentially influenced by biological, behavioural and environmental factors. To investigate the importance of individual-level and school-level characteristics in explaining variation in children's physical fitness. The sample comprised 354 Portuguese children, aged 5-10 years. Physical fitness [(handgrip (HG) strength, standing long jump (SLJ), shuttle-run (SR), total physical fitness score (PFz)], gross motor coordination (GMC) and body mass index (BMI) were assessed. Moderate-to-vigorous physical activity (MVPA) was objectively monitored; birth weight and socio-economic status (SES) were obtained by questionnaire. School contexts were assessed via an objective audit. Multilevel models using the Bayesian approach were used. Age was positively associated with HG, SLJ and PFz (p < 0.05); BMI was positively associated with HG (p < 0.05) and GMC with all physical fitness components (p < 0.05); MVPA was only associated with PFz (p < 0.05); children with level A SES outperformed those with level C SES in HG (p < 0.05), but those with level B SES outperformed their level A SES peers in SR (p < 0.05). Within the school context, no equipment for physical education was negatively associated with HG (p < 0.05). In the primary school years, individual-level characteristics were more important than the school context in explaining variations in physical fitness.


Vascular disruption in combination with mTOR inhibition in renal cell carcinoma.
Renal cell carcinoma (RCC) is an angiogenesis-dependent and hypoxia-driven malignancy. As a result, there has been an increased interest in the use of antiangiogenic agents for the management of RCC in patients. However, the activity of tumor-vascular disrupting agents (tumor-VDA) has not been extensively examined against RCC. In this study, we investigated the therapeutic efficacy of the tumor-VDA ASA404 (DMXAA, 5,6-dimethylxanthenone-4-acetic acid, or vadimezan) in combination with the mTOR inhibitor everolimus (RAD001) against RCC. In vitro studies were carried out using human umbilical vein endothelial cells and in vivo studies using orthotopic RENCA tumors and immunohistochemical patient tumor-derived RCC xenografts. MRI was used to characterize the vascular response of orthotopic RENCA xenografts to combination treatment. Therapeutic efficacy was determined by tumor growth measurements and histopathologic evaluation. ASA404/everolimus combination resulted in enhanced inhibition of endothelial cell sprouting in the 3-dimensional spheroid assay. MRI of orthotopic RENCA xenografts revealed an early increase in permeability 4 hours posttreatment with ASA404, but not with everolimus. Twenty-four hours after treatment, a significant reduction in blood volume was observed with combination treatment. Correlative CD31/NG2 staining of tumor sections confirmed marked vascular damage following combination therapy. Histologic sections showed extensive necrosis and a reduction in the viable rim following combination treatment compared with VDA treatment alone. These results show the potential of combining tumor-VDAs with mTOR inhibitors in RCC. Further investigation into this novel combination strategy is warranted.


Hexane Extract of Orthosiphon stamineus Induces Insulin Expression and Prevents Glucotoxicity in INS-1 Cells.
Hyperglycemia, a characteristic feature of diabetes, induces glucotoxicity in pancreatic β-cells, resulting in further impairment of insulin secretion and worsening glycemic control. Thus, preservation of insulin secretory capacity is essential for the management of type 2 diabetes. In this study, we evaluated the ability of an Orthosiphon stamineus (OS) extract to prevent glucotoxicity in insulin-producing cells. We measured insulin mRNA expression and glucose-stimulated insulin secretion (GSIS) in OS-treated INS-1 cells after exposure to a high glucose (HG; 30 mM) concentration. The hexane extract of OS elevated mRNA expression of insulin as well as pancreatic and duodenal homeobox-1 of INS-1 cells in a dose-dependent manner. The hexane OS extract also increased the levels of phosphorylated phosphatidylinositol 3-kinase (PI3K) in a concentration-dependent manner. Additionally, Akt phosphorylation was elevated by treatment with 100 and 200 µmol of the hexane OS extract. Three days of HG exposure suppressed insulin mRNA expression and GSIS; these expressions were restored by treatment with the hexane OS extract. HG elevated peroxide levels in the INS-1 cells. These levels were unaffected by OS treatment under both normal and hyperglycemic conditions. Our results suggested that the hexane extract of OS elevates insulin mRNA expression and prevents glucotoxicity induced by a 3-day treatment with HG. This was associated with the activation of PI-3K and Akt.


Amphiregulin activates human hepatic stellate cells and is upregulated in non alcoholic steatohepatitis.
Amphiregulin (AR) involvement in liver fibrogenesis and hepatic stellate cells (HSC) regulation is under study. Non-alcoholic fatty liver disease (NAFLD) and its more severe form non-alcoholic steatohepatitis (NASH) may progress to cirrhosis and hepatocellular cancer (HCC). Our aim was to investigate ex vivo the effect of AR on human primary HSC (hHSC) and verify in vivo the relevance of AR in NAFLD fibrogenesis. hHSC isolated from healthy liver segments were analyzed for expression of AR and its activator, TNF-α converting enzyme (TACE). AR induction of hHSC proliferation and matrix production was estimated in the presence of antagonists. AR involvement in fibrogenesis was also assessed in a mouse model of NASH and in humans with NASH. hHSC time dependently expressed AR and TACE. AR increased hHSC proliferation through several mitogenic signaling pathways such as EGFR, PI3K and p38. AR also induced marked upregulation of hHSC fibrogenic markers and reduced hHSC death. AR expression was enhanced in the HSC of a murine model of NASH and of severe human NASH. In conclusion, AR induces hHSC fibrogenic activity via multiple mitogenic signaling pathways, and is upregulated in murine and human NASH, suggesting that AR antagonists may be clinically useful anti-fibrotics in NAFLD.


Postnatal development of intrinsic and synaptic properties transforms signaling in the layer 5 excitatory neural network of the visual cortex.
Information flow in neocortical circuits is regulated by two key parameters: intrinsic neuronal properties and the short-term activity-dependent plasticity of synaptic transmission. Using multineuronal whole-cell voltage recordings, we characterized the postnatal maturation of the electrophysiological properties and short-term plasticity of excitatory synaptic transmission between pairs of layer 5 (L5) pyramidal neurons (n = 158) in acute slices of rat visual cortex over the first postnatal month. We found that the intrinsic and synaptic properties of L5 pyramidal neurons develop in parallel. Before postnatal day 15 (P15), intrinsic electrophysiological properties were tuned to low-frequency operation, characterized by high apparent input resistance, a long membrane time constant, and prolonged somatic action potentials. Unitary excitatory synaptic potentials were of large amplitude (P11-P15; median, 514 μV), but showed pronounced use-dependent depression during prolonged regular and physiologically relevant presynaptic action potential firing patterns. In contrast, in mature animals we observed a developmental decline of the peak amplitude of unitary EPSPs (P25-P29; median, 175 μV) paralleled by a decrease in apparent input resistance, membrane time constant, and somatic action potential duration. Notably, synaptic signaling of complex action potential firing patterns was also transformed, with P25-P29 connections faithfully signaling action potential trains at frequencies up to 40 Hz (1st to 50th action potential ratio, 0.91 ± 0.12). Postnatal refinement of intrinsic properties and short-term plasticity therefore transforms the capacity of the L5 excitatory neural network of the visual cortex to generate and process patterns of action potential firing and contribute to network activity.


Pump-linewidth-tolerant optical wavelength conversion for high-order QAM signals using coherent pumps.
Optical wavelength conversion (OWC) is expected to be a desirable function in future optical transparent networks. Since high-order quadrature amplitude modulation (QAM) is more sensitive to the phase noise, in the OWC of high-order QAM signals, it is crucial to suppress the extra noise introduced in the OWC subsystem, especially for the scenario with multiple cascaded OWCs. Here, we propose and experimentally demonstrate a pump-linewidth-tolerant OWC scheme suitable for high-order QAM signals using coherent two-tone pumps. Using 3.5-MHz-linewidth distributed feedback (DFB) lasers as pump sources, our scheme enables wavelength conversion of both 16QAM and 64QAM signals with negligible power penalty, in a periodically-poled Lithium Niobate (PPLN) waveguide based OWC. We also demonstrate the performance of pump phase noise cancellation, showing that such coherent two-tone pump schemes can eliminate the need for ultra-narrow linewidth pump lasers and enable practical implementation of low-cost OWC in future dynamic optical networks.


Understanding medical care of transplant recipients through integrated registry and pharmacy claims data.
Limited data exist on medication use aside from immunosuppression among large samples of kidney transplant recipients. We examined a novel database wherein Organ Procurement and Transplantation Network (OPTN) registry data were linked to records from a US pharmaceutical claims clearinghouse (2005-2010 claims) to examine pharmaceutical care at the first transplant anniversary (n = 16,157). We quantified the use of the following medication types within ±60 days of the first-year OPTN report according to estimated glomerular filtration rate (eGFR): antihypertensives, lipid-lowering, bone and mineral, and anemia treatments. Adjusted associations of medication use with eGFR and other clinical factors were quantified by multivariate logistic regression. Requirements for multiple antihypertensive agents rose with lower eGFR, with β-blockers comprising the most commonly used antihypertensive agent. The adjusted likelihood of vitamin D (adjusted odds ratio (aOR) 2.07, 95% CI 1.19-3.59) and especially erythrocyte-stimulating agents (aOR 19.94, 95% CI 7.01-56.00) rose in a graded manner to peak with eGFR <15 versus >90, whereas statin use was most common with eGFR 30-59 ml/min/1.73 m(2). Black race was independently associated with increased use of all classes of antihypertensives and vitamin D, but lower adjusted statin use. Rapamycin-based immunosuppression was associated with increased use of statins and erythrocyte-stimulating agents. Integrated registry and pharmacy fill data provide a novel tool for pharmacoepidemiologic investigations of delivered post-transplant care. Transplant recipients with reduced renal function have increased requirements for pharmaceutical care of comorbidities. Causes of racial variation in medication fills warrant further investigation.


Enhancing S-adenosyl-methionine catabolism extends Drosophila lifespan.
Methionine restriction extends the lifespan of various model organisms. Limiting S-adenosyl-methionine (SAM) synthesis, the first metabolic reaction of dietary methionine, extends longevity in Caenorhabditis elegans but accelerates pathology in mammals. Here, we show that, as an alternative to inhibiting SAM synthesis, enhancement of SAM catabolism by glycine N-methyltransferase (Gnmt) extends the lifespan in Drosophila. Gnmt strongly buffers systemic SAM levels by producing sarcosine in either high-methionine or low-sams conditions. During ageing, systemic SAM levels in flies are increased. Gnmt is transcriptionally induced in a dFoxO-dependent manner; however, this is insufficient to suppress SAM elevation completely in old flies. Overexpression of gnmt suppresses this age-dependent SAM increase and extends longevity. Pro-longevity regimens, such as dietary restriction or reduced insulin signalling, attenuate the age-dependent SAM increase, and rely at least partially on Gnmt function to exert their lifespan-extending effect in Drosophila. Our study suggests that regulation of SAM levels by Gnmt is a key component of lifespan extension.


Quantification of proteins using peptide immunoaffinity enrichment coupled with mass spectrometry.
There is a great need for quantitative assays in measuring proteins. Traditional sandwich immunoassays, largely considered the gold standard in quantitation, are associated with a high cost, long lead time, and are fraught with drawbacks (e.g. heterophilic antibodies, autoantibody interference, 'hook-effect').(1) An alternative technique is affinity enrichment of peptides coupled with quantitative mass spectrometry, commonly referred to as SISCAPA (Stable Isotope Standards and Capture by Anti-Peptide Antibodies).(2) In this technique, affinity enrichment of peptides with stable isotope dilution and detection by selected/multiple reaction monitoring mass spectrometry (SRM/MRM-MS) provides quantitative measurement of peptides as surrogates for their respective proteins. SRM/MRM-MS is well established for accurate quantitation of small molecules (3, 4) and more recently has been adapted to measure the concentrations of proteins in plasma and cell lysates.(5-7) To achieve quantitation of proteins, these larger molecules are digested to component peptides using an enzyme such as trypsin. One or more selected peptides whose sequence is unique to the target protein in that species (i.e. "proteotypic" peptides) are then enriched from the sample using anti-peptide antibodies and measured as quantitative stoichiometric surrogates for protein concentration in the sample. Hence, coupled to stable isotope dilution (SID) methods (i.e. a spiked-in stable isotope labeled peptide standard), SRM/MRM can be used to measure concentrations of proteotypic peptides as surrogates for quantification of proteins in complex biological matrices. The assays have several advantages compared to traditional immunoassays. The reagents are relatively less expensive to generate, the specificity for the analyte is excellent, the assays can be highly multiplexed, enrichment can be performed from neat plasma (no depletion required), and the technique is amenable to a wide array of proteins or modifications of interest.(8-13) In this video we demonstrate the basic protocol as adapted to a magnetic bead platform.


Pancreatic polypeptide is recognized by two hydrophobic domains of the human Y4 receptor binding pocket.
Structural characterization of the human Y4 receptor (hY4R) interaction with human pancreatic polypeptide (hPP) is crucial, not only for understanding its biological function but also for testing treatment strategies for obesity that target this interaction. Here, the interaction of receptor mutants with pancreatic polypeptide analogs was studied through double-cycle mutagenesis. To guide mutagenesis and interpret results, a three-dimensional comparative model of the hY4R-hPP complex was constructed based on all available class A G protein-coupled receptor crystal structures and refined using experimental data. Our study reveals that residues of the hPP and the hY4R form a complex network consisting of ionic interactions, hydrophobic interactions, and hydrogen binding. Residues Tyr(2.64), Asp(2.68), Asn(6.55), Asn(7.32), and Phe(7.35) of Y4R are found to be important in receptor activation by hPP. Specifically, Tyr(2.64) interacts with Tyr(27) of hPP through hydrophobic contacts. Asn(7.32) is affected by modifications on position Arg(33) of hPP, suggesting a hydrogen bond between these two residues. Likewise, we find that Phe(7.35) is affected by modifications of hPP at positions 33 and 36, indicating interactions between these three amino acids. Taken together, we demonstrate that the top of transmembrane helix 2 (TM2) and the top of transmembrane helices 6 and 7 (TM6-TM7) form the core of the peptide binding pocket. These findings will contribute to the rational design of ligands that bind the receptor more effectively to produce an enhanced agonistic or antagonistic effect.


Influence of fluctuations of plasma large neutral amino acids with normal diets on the clinical response to levodopa.
Plasma large neutral amino acids (LNAAs) compete with levodopa for entry into the brain. Fluctuations in plasma LNAA concentrations could therefore contribute to variability in clinical response to levodopa. The hourly plasma levodopa, plasma LNAAs and clinical response were investigated in 11 fluctuating Parkinsonian patients on a regular hospital diet. The fluctuations in plasma levodopa were 2 to 3 times greater than the fluctuations of plasma LNAAs. The correlation between clinical response and plasma levodopa was substantially improved in only one patient by considering plasma LNAAs and calculating relative levodopa flux into brain. Although plasma LNAAs significantly increased during the day, the patients' clinical status did not uniformly deteriorate and mean afternoon clinical scores correlated better with mean plasma levodopa and levodopa flux than with mean plasma LNAAs. Minimum effective concentrations of levodopa for clinical response did not correlate with 9 am LNAA concentrations. It is concluded that in most patients, the relatively small variation in plasma LNAAs in comparison with the large variations in plasma levodopa indicates that fluctuations in LNAA are not an important contributor to the fluctuating response to levodopa.


Coexistent hereditary and inflammatory neuropathy.
Classically, the course of Charcot-Marie-Tooth (CMT) disease is gradually progressive. We describe eight atypical patients who developed acute or subacute deterioration. Seven of these had genetically proven CMT disease type 1A (CMT1A) due to chromosome 17p11.2-12 duplication, and one had X-linked disease (CMTX) due to a mutation in the GJB1 gene. In this group there was sufficient clinical, electrophysiological and neuropathological information to indicate a diagnosis of a superimposed inflammatory polyneuropathy. The age range of the patients was 18-69 years, with a mean of 39 years. A family history of a similar neuropathic condition was present in only four patients. All eight had an acute or subacute deterioration following a long asymptomatic or stable period. Seven had neuropathic pain or prominent positive sensory symptoms. Nerve biopsy demonstrated excess lymphocytic infiltration in all eight patients. Five patients were treated with steroids and/or intravenous immunoglobulin, with variable positive response; three patients received no immunomodulatory treatment. Inflammatory neuropathy has previously been recognized in patients with hereditary neuropathy, with uncharacterized genetic defects and with CMT1B. We present detailed assessments of patients with CMT1A and CMTX, including nerve biopsy, and conclude that coexistent inflammatory neuropathy is not genotype-specific in hereditary motor and sensory neuropathy. Although this was not a formal epidemiological study, estimates of the prevalence of CMT disease and chronic inflammatory demyelinating polyneuropathy indicate that the association is more frequent than would be expected by chance. This has implications for understanding the pathogenesis of inflammatory neuropathies and raises important considerations in the management of patients with hereditary neuropathies. If a patient with CMT disease experiences an acute or subacute deterioration in clinical condition, treatment of a coexistent inflammatory neuropathy with steroids or immunoglobulin should be considered.


Y-chromosome evidence supports asymmetric dog introgression into eastern coyotes.
Hybridization has played an important role in the evolutionary history of Canis species in eastern North America. Genetic evidence of coyote-dog hybridization based on mitochondrial DNA (mtDNA) is lacking compared to that based on autosomal markers. This discordance suggests dog introgression into coyotes has potentially been male biased, but this hypothesis has not been formally tested. Therefore, we investigated biparentally, maternally, and paternally inherited genetic markers in a sample of coyotes and dogs from southeastern Ontario to assess potential asymmetric dog introgression into coyotes. Analysis of autosomal microsatellite genotypes revealed minimal historical and contemporary admixture between coyotes and dogs. We observed only mutually exclusive mtDNA haplotypes in coyotes and dogs, but we observed Y-chromosome haplotypes (Y-haplotypes) in both historical and contemporary coyotes that were also common in dogs. Species-specific Zfy intron sequences of Y-haplotypes shared between coyotes and dogs confirmed their homology and indicated a putative origin from dogs. We compared Y-haplotypes observed in coyotes, wolves, and dogs profiled in multiple studies, and observed that the Y-haplotypes shared between coyotes and dogs were either absent or rare in North American wolves, present in eastern coyotes, but absent in western coyotes. We suggest the eastern coyote has experienced asymmetric genetic introgression from dogs, resulting from predominantly historical hybridization with male dogs and subsequent backcrossing of hybrid offspring with coyotes. We discuss the temporal and spatial dynamics of coyote-dog hybridization and the conditions that may have facilitated the introgression of dog Y-chromosomes into coyotes. Our findings clarify the evolutionary history of the eastern coyote.