The utility of t(14;18) in understanding risk factors for non-Hodgkin lymphoma.
Characteristic chromosomal abnormalities are associated with specific histological subtypes of non-Hodgkin lymphoma (NHL). The chromosomal translocation t(14;18)(q32;q21) is one of the most common chromosomal abnormalities in NHL, occurring in 70%-90% of cases of follicular lymphoma, 20%-30% of diffuse large B-cell lymphoma, and 5%-10% of other less common subtypes. The t(14;18)-positive NHL may represent a homogenous group and, consequently, increase etiologic specificity in epidemiological studies. Although the t(14;18) has important clinical ramifications, its etiologic significance remains to be determined. Two population-based, case-control studies addressed this issue by evaluating potential risk factors for t(14;18)-positive and t(14;18)-negative subgroups of NHL. Both studies found that the association between pesticide exposures and risk of NHL was largely limited to t(14;18)-positive NHL cases. However, the findings regarding cigarette smoking, family history of hematopoietic cancer, and hair dye use were not entirely consistent. These results indicate that defining subgroups of NHL according to t(14;18) status may be useful for etiologic research, particularly for exposures that are genotoxic or may contribute to the development of NHL through pathways involving the t(14;18). Studies to further evaluate these associations and delineate the effects of various exposures in other genetically defined subgroups of NHL are warranted.


Associations Between Methotrexate Use and the Risk of Cardiovascular Events in Patients with Elderly-onset Rheumatoid Arthritis.
We evaluated the associations between time-varying methotrexate (MTX) use and risk of cardiovascular events (CVE) in patients with rheumatoid arthritis (RA). We studied an inception cohort of 23,994 patients with RA diagnosed after their 65th birthday. Multivariable Cox regression models were fit to evaluate the associations between time-varying MTX use, controlling for other risk factors, and time to CVE. Alternative models assessed the cumulative duration of MTX use over the (1) first year, (2) previous year (recent use), and (3) entire duration of followup. We also assessed whether the strength of the association varied over time. Over 115,453 patient-years (PY), 3294 (13.7%) patients experienced a CVE (28.5 events per 1000 PY; 95% CI 27.6-29.5). In the multivariable analyses, the model assessing time-varying continuous use in the most recent year yielded the best fit. Increasing recent MTX use was associated with lower CVE risks (HR 0.79 for continuous use vs no use in past 12 months, 95% CI 0.70-0.88; p < 0.0001). Greater MTX use in the first year after cohort entry was also protective (HR 0.84, 95% CI 0.72-0.96; p = 0.0048), but this effect decreased with increasing followup. In contrast, longer MTX use during the entire followup was not clearly associated with CVE risk (HR 0.98, 95% CI 0.95-1.01; p = 0.1441). We observed about a 20% decrease in CVE associated with recent continuous MTX use. Greater MTX use in the first year of cohort entry also appeared to be important in the association between MTX and CVE risk.


WE-G-217A-03: Respiratory-Related External/Internal Motion Based MR Image Reconstruction Using Dynamic Keyhole for Real-Time Tumor Monitoring.
It is aim to test the hypothesis that a dynamic keyhole MRI reconstruction technique using external/internal respiratory surrogate position reduces acquisition time while retaining image quality for real-time tumor monitoring, compared to the conventional keyhole technique. 46 thoracic MRI studies with 13 healthy human subjects have been acquired using a 3T GE MRI. Acquired MR images were reconstructed using zero-filling, conventional keyhole and the proposed respiratory motion based dynamic keyhole techniques; resultant images were then compared for image quality. Undersampled k-space rate in the phase encoding direction was determined based on the difference between the original image and the reconstructed image. The position of abdominal muscles and diaphragm were used to determine any excess data that exists in the overlaid temporal data. In addition, the feasibility of the dynamic keyhole method was applied using lung tumor MR images. The result from dynamic keyhole using respiratory motion demonstrated significant improvement compared with the zero-filling and conventional keyhole methods. Firstly, the dynamic keyhole method using external respiratory motion had an overlaid average of 79.7% (204 lines) of 256 lines from 46 datasets, compared to 63.9% in zero-filling and 74.3% in conventional keyhole. Secondly, dynamic keyhole method using internal diaphragm motion had an overlaid average of 84.5% (216 lines) of 256 lines from all datasets compared to 67.1% and 77.8% or zero-filling and conventional keyhole, respectively. Lastly, dynamic keyhole has been validated with one dataset involving lung tumor MR images. Image blurring artifacts and inferior resolution were not present in the final MR images using dynamic keyhole. Dynamic keyhole method using respiratory external/internal surrogate motion has been proposed to reconstruct MR images without image artifacts and with superior resolution. This method is applicable to MR images targeting lungs and other organs affected by respiratory motion for real-time tumor motion monitoring.


Prevalence, types and demographic features of child labour among school children in Nigeria.
BACKGROUND: To determine the prevalence, types and demographic features of child labour among school children in Nigeria. METHODS: A cross-sectional interview study of 1675 randomly selected public primary and secondary school pupils aged 5 to less than 18 years was conducted in the Sagamu Local Government Area of Ogun State, Nigeria from October 1998 to September 1999. RESULTS: The overall prevalence of child labour was 64.5%: 68.6% among primary and 50.3% among secondary school pupils. Major economic activities included street trading (43.6%), selling in kiosks and shops (25.4%) and farming (23.6%). No child was involved in bonded labour or prostitution. Girls were more often involved in labour activities than boys (66.8% versus 62.1%, p = 0.048): this difference was most obvious with street trading (p = 0.0004). Most of the children (82.2%) involved in labour activities did so on the instruction of one or both parents in order to contribute to family income. Children of parents with low socio-economic status or of poorly educated parents were significantly involved in labour activities (p = 0.01 and p = 0.001 respectively). Child labour was also significantly associated with increasing number of children in the family size (p = 0.002). A higher prevalence rate of child labour was observed among children living with parents and relations than among those living with unrelated guardians. CONCLUSION: It is concluded that smaller family size, parental education and family economic enhancement would reduce the pressure on parents to engage their children in labour activities.


Evaluation of immunogenicity of an oral Salmonella vaccine expressing recombinant Plasmodium berghei merozoite surface protein-1.
A repetitive region of Plasmodium berghei merozoite surface protein-1 (PbMSP-1) was expressed as a fusion protein with either maltose binding protein or the B subunit of heat-labile enterotoxin from Escherichia coli. Vaccination of mice with the fusion proteins indicates that this region of PbMSP-1 is antigenic as evidenced by an antibody response. The fusion proteins were also expressed in Salmonella and mice were orally immunized with the recombinant Salmonella. Some of the vaccinated mice survived a challenge with P. berghei blood-stage parasites without developing parasitemia. All control mice became patent and succumbed to the challenge infection. This partial protection was also observed with purified recombinant protein and was independent of the adjuvant used. Mice immunized with recombinant Salmonella showed either extremely low or no antibody response to PbMSP-1, suggesting that cell-mediated immunity is important for the observed protection. These studies show that it is feasible to develop a cost effective oral vaccine against the blood stage of the malarial parasite.


Electrochemical live monitoring of tumor cell migration out of micro-tumors on an innovative multiwell high-dense microelectrode array.
Understanding of cell migration and spreading out of tumor tissue is of great interest concerning the mechanism and causes of tumor malignancy and metastases. Although there are methods available for studying cell migration on monolayer cell cultures like transwell assays, novel techniques for monitoring cell spreading out of 3D organoids or tumor tissue samples are highly required. In this context, we developed an innovative high-dense microelectrode array for impedimetric monitoring of cell migration from 3D tumor cultures. For a proof of concept, a strongly migrating breast cancer cell line (MDA-MB-231) and two malignant melanoma cell lines (T30.6.9, T12.8.10ZII) were used for generating viable micro-tumor models. The migration propensity was determined by impedimetric monitoring over 144 hours, correlated by microscopy and validated by transwell assays. The impedimetric analysis of covered electrodes and the relative impedance maximum values revealed extended information regarding the contribution of proliferative effects. More strikingly, using reference populations of mitomycin C treated spheroids where proliferation was suppressed, distinction of proliferation and migration was possible. Therefore, our high-dense microelectrode array based impedimetric migration monitoring has the capability for an automated quantitative analysis system that can be easily scaled up as well as integrated in lab on chip devices.


IL-21 Biased Alemtuzumab Induced Chronic Antibody-Mediated Rejection Is Reversed by LFA-1 Costimulation Blockade.
Despite its excellent efficacy in controlling T cell mediated acute rejection, lymphocyte depletion may promote a humoral response. While T cell repopulation after depletion has been evaluated in many aspects, the B cell response has not been fully elucidated. We tested the hypothesis that the mechanisms also involve skewed T helper phenotype after lymphocytic depletion. Post-transplant immune response was measured from alemtuzumab treated hCD52Tg cardiac allograft recipients with or without anti-LFA-1 mAb. Alemtuzumab induction promoted serum DSA, allo-B cells, and CAV in humanized CD52 transgenic (hCD52Tg) mice after heterotopic heart transplantation. Additional anti-LFA-1 mAb treatment resulted in reduced DSA (Fold increase 4.75 ± 6.9 vs. 0.7 ± 0.5; p < 0.01), allo-specific B cells (0.07 ± 0.06 vs. 0.006 ± 0.002 %; p < 0.01), neo-intimal hyperplasia (56 ± 14% vs. 23 ± 13%; p < 0.05), arterial disease (77.8 ± 14.2 vs. 25.8 ± 20.1%; p < 0.05), and fibrosis (15 ± 23.3 vs. 4.3 ± 1.65%; p < 0.05) in this alemtuzumab-induced chronic antibody-mediated rejection (CAMR) model. Surprisingly, elevated serum IL-21 levels in alemtuzumab-treated mice was reduced with LFA-1 blockade. In accordance with the increased serum IL-21 level, alemtuzumab treated mice showed hyperplastic germinal center (GC) development, while the supplemental anti-LFA-1 mAb significantly reduced the GC frequency and size. We report that the incomplete T cell depletion inside of the GC leads to a systemic IL-21 dominant milieu with hyperplastic GC formation and CAMR. Conventional immunosuppression, such as tacrolimus and rapamycin, failed to reverse AMR, while co-stimulation blockade with LFA-1 corrected the GC hyperplastic response. The identification of IL-21 driven chronic AMR elucidates a novel mechanism that suggests a therapeutic approach with cytolytic induction.


Mitofusin 2 is necessary for transport of axonal mitochondria and interacts with the Miro/Milton complex.
Mitofusins (Mfn1 and Mfn2) are outer mitochondrial membrane proteins involved in regulating mitochondrial dynamics. Mutations in Mfn2 cause Charcot-Marie-Tooth disease (CMT) type 2A, an inherited disease characterized by degeneration of long peripheral axons, but the nature of this tissue selectivity remains unknown. Here, we present evidence that Mfn2 is directly involved in and required for axonal mitochondrial transport, distinct from its role in mitochondrial fusion. Live imaging of neurons cultured from Mfn2 knock-out mice or neurons expressing Mfn2 disease mutants shows that axonal mitochondria spend more time paused and undergo slower anterograde and retrograde movements, indicating an alteration in attachment to microtubule-based transport systems. Furthermore, Mfn2 disruption altered mitochondrial movement selectively, leaving transport of other organelles intact. Importantly, both Mfn1 and Mfn2 interact with mammalian Miro (Miro1/Miro2) and Milton (OIP106/GRIF1) proteins, members of the molecular complex that links mitochondria to kinesin motors. Knockdown of Miro2 in cultured neurons produced transport deficits identical to loss of Mfn2, indicating that both proteins must be present at the outer membrane to mediate axonal mitochondrial transport. In contrast, disruption of mitochondrial fusion via knockdown of the inner mitochondrial membrane protein Opa1 had no effect on mitochondrial motility, indicating that loss of fusion does not inherently alter mitochondrial transport. These experiments identify a role for mitofusins in directly regulating mitochondrial transport and offer important insight into the cell type specificity and molecular mechanisms of axonal degeneration in CMT2A and dominant optic atrophy.


Tractography dissection variability: What happens when 42 groups dissect 14 white matter bundles on the same dataset?
White matter bundle segmentation using diffusion MRI fiber tractography has become the method of choice to identify white matter fiber pathways in vivo in human brains. However, like other analyses of complex data, there is considerable variability in segmentation protocols and techniques. This can result in different reconstructions of the same intended white matter pathways, which directly affects tractography results, quantification, and interpretation. In this study, we aim to evaluate and quantify the variability that arises from different protocols for bundle segmentation. Through an open call to users of fiber tractography, including anatomists, clinicians, and algorithm developers, 42 independent teams were given processed sets of human whole-brain streamlines and asked to segment 14 white matter fascicles on six subjects. In total, we received 57 different bundle segmentation protocols, which enabled detailed volume-based and streamline-based analyses of agreement and disagreement among protocols for each fiber pathway. Results show that even when given the exact same sets of underlying streamlines, the variability across protocols for bundle segmentation is greater than all other sources of variability in the virtual dissection process, including variability within protocols and variability across subjects. In order to foster the use of tractography bundle dissection in routine clinical settings, and as a fundamental analytical tool, future endeavors must aim to resolve and reduce this heterogeneity. Although external validation is needed to verify the anatomical accuracy of bundle dissections, reducing heterogeneity is a step towards reproducible research and may be achieved through the use of standard nomenclature and definitions of white matter bundles and well-chosen constraints and decisions in the dissection process.


Molecular detection and isolation from antarctica of methylotrophic bacteria able to grow with methylated sulfur compounds.
This study is the first demonstration that a diverse facultatively methylotrophic microbiota exists in some Antarctic locations. PCR amplification of genes diagnostic for methylotrophs was carried out with bacterial DNA isolated from 14 soil and sediment samples from ten locations on Signy Island, South Orkney Islands, Antarctica. Genes encoding the mxaF of methanol dehydrogenase, the fdxA for Afipia ferredoxin, the msmA of methanesulfonate monooxygenase, and the 16S rRNA gene of Methylobacterium were detected in all samples tested. The mxaF gene sequences corresponded to those of Hyphomicrobium, Methylobacterium, and Methylomonas. Over 30 pure cultures of methylotrophs were isolated on methanesulfonate, dimethylsulfone, or dimethylsulfide from ten Signy Island lakes. Some were identified from 16S rRNA gene sequences (and morphology) as Hyphomicrobium species, strains of Afipia felis, and a methylotrophic Flavobacterium strain. Antarctic environments thus contain diverse methylotrophic bacteria, growing on various C1-substrates, including C1-sulfur compounds.


Construction of a self-complementary nucleoside from deoxyguanosine.
The 8-hydroxyguanine:adenine mispairing scheme that spontaneously occurs in vivo through oxidative metabolism of DNA was edited to obtain a pair closely fitting the Watson-Crick geometry in which 2 purine bases of identical structure but oppositely rotated in the syn and anti configurations are hydrogen-bonded. The structure thus designed, 2-amino-8-hydroxypurine, was synthesized as a DNA precursor from deoxyguanosine in 8 steps by oxidation at carbon 8 of guanine followed by reduction at carbon 6. Polydeoxynucleotides embodying this self-complementary base are expected to undergo direct copying processes through polymerase catalysis.


European continuing medical education in vascular surgery: 5-year results of congresses approved by the Union Européenne des Médecins Spécialistes Section of Vascular Surgery.
Continuing medical education (CME) can be defined as ''educational activities that serve to maintain, develop, or increase the knowledge, skills and professional performance of a physician to provide services for patients, the public, or the profession''. CME is a major professional responsibility. The European Board of Vascular Surgery of the Union Européenne des Médecins Spécialistes (UEMS) Section of Vascular Surgery has, through its European Vascular CME (EVCME) Committee, accredited 74 congresses during the 5-year period from 2000-2004. Official evaluation forms were completed by the congress participants for a personal appraisal of the quality of the activities. The data in this manuscript focused on questions that were the most relevant and of the greatest interest to the participants. A statistical analysis of the results was performed utilizing ANOVA and Robust tests of equality of means as well as a posthoc analysis for further investigation, and non parametric Wilcoxon signed ranks test. The educational needs of participants regarding new diagnostic and therapeutic modes were stated as ''important'' and ''extremely important'' in the responses at over 80% in total. Over 75% of the participants answered ''extremely important'' and ''important'' to the question ''how important is evidence-based practice to your practice''. This survey indicates that the EVCME approved congresses had a positive impact for the vascular surgeon by updating overall knowledge on vascular surgery; the majority of comments by the participants also indicates that EVCME is fulfilling its aim to bring as much evidence-based practice as possible into the daily work schedule of the surgeon by turning knowledge acquired by CME into performance of the participants.


AIE nanodots scaffolded by mini-ferritin protein for cellular imaging and photodynamic therapy.
Photodynamic therapy (PDT) is one of the most elegant cancer treatment strategies that can be controlled by a beam of light with non-invasion, precise control, and high spatiotemporal accuracy. An ideal photosensitizer (PS) is the key to ensure the efficacy of PDT. Due to their hydrophobic and rigid planar structures, most traditional PSs are prone to aggregate under physiological conditions, which causes fluorescence quenching and significantly reduces reactive oxygen species (ROS) generation. Fortunately, the emergence of aggregation-induced emission (AIE) dyes offers a potential opportunity to overcome these limitations. When AIE PS molecules are in the aggregation state, the fluorescence intensity and ROS production can be increased. We herein use red AIE PS molecules to prepare stable AIE nanodots for cell imaging and PDT via a simple method with a highly negatively charged mini-ferritin protein as the scaffold. The as-prepared protein-AIE nanodots show strong fluorescence emission and efficient singlet oxygen generation, with good stability, relatively long wavelengths of absorption and emission, and negligible dark toxicity. The mini-ferritin-AIE system may be useful in developing novel functional probes for tumour nanotheranostics.


Synaptic estrogen receptor-alpha levels in prefrontal cortex in female rhesus monkeys and their correlation with cognitive performance.
In rat hippocampus, estrogen receptor-α (ER-α) can initiate nongenomic signaling mechanisms that modulate synaptic plasticity in response to either circulating or locally synthesized estradiol (E). Here we report quantitative electron microscopic data demonstrating that ER-α is present within excitatory synapses in dorsolateral prefrontal cortex (dlPFC) of young and aged ovariectomized female rhesus monkeys with and without E treatment. There were no treatment or age effects on the percentage of excitatory synapses containing ER-α, nor were there any group differences in distribution of ER-α within the synapse. However, the mean size of synapses containing ER-α was larger than that of unlabeled excitatory synapses. All monkeys were tested on delayed response (DR), a cognitive test of working memory that requires dlPFC. In young ovariectomized monkeys without E treatment, presynaptic ER-α correlated with DR accuracy across memory delays. In aged monkeys that received E treatment, ER-α within the postsynaptic density (30-60 nm from the synaptic membrane) positively correlated with DR performance. Thus, although the lack of group effects suggests that ER-α is primarily in synapses that are stable across age and treatment, synaptic abundance of ER-α is correlated with individual performance in two key age/treatment groups. These data have important implications for individual differences in the cognitive outcome among menopausal women and promote a focus on cortical estrogen receptors for therapeutic efficacy with respect to cognition.


Extracted oat and barley β-glucans do not affect cholesterol metabolism in young healthy adults.
β-Glucans are known to exhibit hypocholesterolemic effects. Increased intestinal viscosity is thought to be crucial for cholesterol lowering. It is suggested that concentration, molecular mass, and structure, including the ratio of (1→3) to (1→4) glucan bonds in the molecule, are of importance for β-glucan functionality. This study investigated the effects of 3 different β-glucan sources, incorporated into a beverage and yogurt, on blood lipids and fecal endpoints. Fourteen participants completed this randomized, crossover, single-blinded study with four 3-wk periods: control and 3.3 g/d oat, barley, and barley mutant β-glucans of similar molecular mass. Before and after each period, fasting and postprandial blood samples were drawn and 3-d fecal samples were collected. Treatment did not affect changes in total, LDL, and HDL cholesterol compared with control; however, consumption of 3.3 g/d of oat β-glucans for 3 wk resulted in greater decreases in total (-0.29 ± 0.09 mmol/L, P < 0.01), LDL (-0.23 ± 0.07 mmol/L, P < 0.01), and HDL (-0.05 ± 0.03 mmol/L, P < 0.05) cholesterol compared with baseline. Changes in LDL in the β-glucan treatments were not related to β-glucan structure (cellotriosyl:cellotetraosyl). Decreases in fasting triacylglycerol were substantially greater after oat β-glucan treatment compared with control (P = 0.03). Fecal dry and wet weight, stool frequency, fecal pH, and energy excretion were unaffected. The results do not fully support the hypocholesterolemic effects by differently structured oat and barley β-glucans. However, substantial differences compared with baseline suggest a potential for oat β-glucan, presumably due to its higher solubility and viscosity. This underlines the importance of elusive structural β-glucan features for beneficial physiologic effects.


Raising fluid walls around living cells.
An effective transformation of the cell culture dishes that biologists use every day into microfluidic devices would open many avenues for miniaturizing cell-based workflows. In this article, we report a simple method for creating microfluidic arrangements around cells already growing on the surface of standard petri dishes, using the interface between immiscible fluids as a "building material." Conventional dishes are repurposed into sophisticated microfluidic devices by reshaping, on demand, the fluid structures around living cells. Moreover, these microfluidic arrangements can be further reconfigured during experiments, which is impossible with most existing microfluidic platforms. The method is demonstrated using workflows involving cell cloning, the selection of a particular clone from among others in a dish, drug treatments, and wound healing. The versatility of the approach and its biologically friendly aspects may hasten uptake by biologists of microfluidics, so the technology finally fulfills its potential.


Stt7-dependent phosphorylation during state transitions in the green alga Chlamydomonas reinhardtii.
Photosynthetic organisms are able to adapt to changes in light conditions by balancing the light excitation energy between the light-harvesting systems of photosystem (PS) II and photosystem I to optimize the photosynthetic yield. A key component in this process, called state transitions, is the chloroplast protein kinase Stt7/STN7, which senses the redox state of the plastoquinone pool. Upon preferential excitation of photosystem II, this kinase is activated through the cytochrome b(6)f complex and required for the phosphorylation of the light-harvesting system of photosystem II, a portion of which migrates to photosystem I (state 2). Preferential excitation of photosystem I leads to the inactivation of the kinase and to dephosphorylation of light-harvesting complex (LHC) II and its return to photosystem II (state 1). Here we compared the thylakoid phosphoproteome of the wild-type strain and the stt7 mutant of Chlamydomonas under state 1 and state 2 conditions. This analysis revealed that under state 2 conditions several Stt7-dependent phosphorylations of specific Thr residues occur in Lhcbm1/Lhcbm10, Lhcbm4/Lhcbm6/Lhcbm8/Lhcbm9, Lhcbm3, Lhcbm5, and CP29 located at the interface between PSII and its light-harvesting system. Among the two phosphorylation sites detected specifically in CP29 under state 2, one is Stt7-dependent. This phosphorylation may play a crucial role in the dissociation of CP29 from PSII and/or in its association to PSI where it serves as a docking site for LHCII in state 2. Moreover, Stt7 was required for the phosphorylation of the thylakoid protein kinase Stl1 under state 2 conditions, suggesting the existence of a thylakoid protein kinase cascade. Stt7 itself is phosphorylated at Ser(533) in state 2, but analysis of mutants with a S533A/D change indicated that this phosphorylation is not required for state transitions. Moreover, we also identified phosphorylation sites that are redox (state 2)-dependent but independent of Stt7 and additional phosphorylation sites that are redox-independent.


Interaction of nimesulide, a cyclooxygenase-2 inhibitor, with cisplatin in normotensive and spontaneously hypertensive rats.
We investigated the effect of administration of nimesulide, a selective cyclooxygenase-2 (COX-2) inhibitor, on cisplatin (CP)-induced nephrotoxicity in rats. WKY rats and SHRs were divided into four groups, each. The first and second groups received saline and oral nimesulide (20mg/kg/day for 6 days), respectively, whereas the third and fourth groups received a single intraperitoneal (i.p.) injection of CP (5mg/kg) and CP (5mg/kg) and nimesulide (20mg/kg/day for 5 days), respectively. At the end of the experiment, rats were anesthetized and blood pressure and renal blood flow (RBF) were monitored, followed by intravenous (i.v.) injection of norepinephrine (NE). Nephrotoxicity was evaluated histopathologically and biochemically. CP caused a reduction in baseline RBF in both WKY and SHRs. It increased the concentrations of urea and creatinine and kidney relative weight, and decreased body weight in both WKY and SHRs. Histopathologically, CP caused remarkable renal damage in both WKY rats and SHRs. Treatment with nimesulide alone did not produce any significant change in any of the above measurements. However, nimesulide aggravated CP-induced renal tissue damage in SHRs, but not in WKY rats. The results show that administration nimesulide augmented the histopathological indices of nephrotoxicity in SHRs, but not in WKY rats.


Systematic cross-selectivity study of the factors influencing template receptor interactions in molecularly imprinted nitrogen heterocycles.
A systematic cross-selectivity study involving a series of structurally related N-methylated and non-methylated substituted pyridines was performed with the aim of evaluating the parameters responsible for template receptor binding in molecularly imprinted polymers. Variation in binding of substrate structure permitted evaluation of the steric restraints of the imprinted cavity. The electrostatic effects, primarily hydrogen-bonding, were investigated through rebinding in chloroform and acetonitrile. All species were non-covalently imprinted in thermoinduced methacrylic acid-ethylene glycol dimethacrylate co-polymers. Evaluation of template properties indicate that a correlation exists between non-specific binding and template basicity for a series of structural isomers. A correlation between non-specific binding and hydrophobicity was also identified for templates increasing in alkyl character. However no overall correlation was observed, as it was speculated that these factors may be competing. All species imprinted, with the exception of 2-dimethylaminopyridine, produced a selective response for the template species. Varying degrees of cross-selectivity were observed for each imprinted polymer. Polymers imprinted with templates of higher basicity demonstrated a greater degree of cross-selectivity relative to those of lower basicity. While overall binding was reduced in acetonitrile relative to chloroform, specificity was increased. This highlights the intrinsic difference in binding sites within imprinted and non-imprinted sites of the polymer. Finally, while the ability of the template species to form a co-operative interaction may be advantageous in producing a selective imprint it is not a prerequisite. For species based on this co-operative interaction the steric environment in the immediate proximity to the binding functionalities are critical to recognition. Steric hindrance of non-functionally active groups can dramatically impair the formation of interactions.


Transmission of CARD15 (NOD2) variants within families of patients with inflammatory bowel disease.
Three single nucleotide polymorphisms (SNPs) in CARD15 have been independently associated with Crohn's disease (CD). Since nothing is known about the transmission of these variants within families, this was the subject of our study in Flemish patients with inflammatory bowel disease (IBD) and their healthy relatives. A cohort of 1,670 individuals (570 CD, 173 UC, 165 healthy controls, 762 first-degree unaffected relatives of CD patients) was genotyped for Arg702Trp, Gly908Arg, and Leu1007fsinsC. Mutant allele and carrier frequencies were compared between groups. Segregation patterns were compared using a bivariate Dale model. The carrier prevalence of CARD15 variants for CD patients was 46.3%, compared to 20.6% for healthy controls and 22.0% for ulcerative colitis (UC) patients (both p < 0.0001). An increased carriage rate of CARD15 variants was observed in unaffected relatives of CD patients (37.3%; p < 0.0001 vs controls), although this was significantly lower than in the CD patients (p = 0.001). Paternal transmission gave a 5.17-fold higher chance for the child to develop the disease compared to maternal transmission (95% CI [1.59, 16.78]; p = 0.0063). UC patients belonging to mixed IBD families carried significantly more mutations (42.3%) compared to other UC patients (18.4%) (p < 0.01). Maternal transmission of the CARD15 variant allele is associated with a lower proportion of affected individuals compared to paternal transmission. Therefore, maternal transmission does not carry an increased risk of transmission as does paternal transmission. The increased mutation carriage in unaffected siblings of CD patients and in UC patients belonging to mixed families suggests that other factors than CARD15 contribute to the eventual disease expression.


Ultrafast nuclear myocardial perfusion imaging on a new gamma camera with semiconductor detector technique: first clinical validation.
To assess the diagnostic performance of a novel ultrafast cardiac gamma camera with cadmium-zinc-telluride (CZT) solid-state semiconductor detectors for nuclear myocardial perfusion imaging (MPI). The study group comprised 75 consecutive patients (55 men, BMI range 19-45 kg/m(2)) who underwent a 1-day (99m)Tc-tetrofosmin adenosine-stress/rest imaging protocol. Scanning was performed first on a conventional dual-detector SPECT gamma camera (Ventri, GE Healthcare) with a 15-min acquisition time each for stress and rest. All scans were immediately repeated on an ultrafast CZT camera (Discovery 530 NMc, GE Healthcare) with a 3-min scan time for stress and a 2-min scan time for rest. Clinical agreement (normal, ischaemia, scar) between CZT and SPECT was assessed for each patient and for each coronary territory using SPECT MPI as the reference standard. Segmental myocardial tracer uptake values (percent of maximum) using a 20-segment model and left ventricular ejection fraction (EF) values obtained using CZT were compared with those obtained using conventional SPECT by intraclass correlation and by calculating Bland-Altman limits of agreement. There was excellent clinical agreement between CZT and conventional SPECT on a per-patient basis (96.0%) and on a per-vessel territory basis (96.4%) as shown by a highly significant correlation between segmental tracer uptake values (r=0.901, p<0.001). Similarly, EF values for both scanners were highly correlated (r=0.976, p<0.001) with narrow Bland-Altman limits of agreement (-5.5-10.6%). The novel CZT camera allows a more than fivefold reduction in scan time and provides clinical information equivalent to conventional standard SPECT MPI.


Association between specific HIV-1 Env traits and virologic control in vivo.
HIV RNA levels are influenced by genetic characteristics of both the host and the virus. Here we applied machine learning techniques to determine if plasma-derived HIV-1 amino acid sequences can be used to predict spontaneous virologic control. We studied the relationship between HIV-1 env genotype and viral load in 20 chronically infected patients undergoing treatment interruptions (SSITT, Swiss-Spanish Intermittent Treatment Trial) and in 104 primary HIV infected (PHI) patients before antiretroviral therapy (cART) and where applicable also after treatment stop. Extensive longitudinal sampling during the interruptions was performed in nine SSITT patients. Sequences obtained from these nine patients during the first virus rebound were used as a training data set and revealed a strong genetic signature (accuracy 98.6% in cross-validation) associated with control of viremia at levels below 5000copies/mL of viral RNA maintained for at least 2 months after the final cART stop. The simple sequence pattern at gp120 positions 268E/358T was confirmed to be predictive of control in the clonal sequences originating from these patients during all subsequent rebounds. Sequences from the remaining 11 SSITT patients with less frequent sampling and from the PHI patients were used for external validation. High sensitivities (71-100%) and negative predictive values (80-100%) but low positive predictive values (12-40%) were achieved in the patient-wise analysis which was based on presence of the genetic pattern in all clones. These results suggest that presence of virus lacking the amino acid pattern 268E/358T is associated with VL >5000 at baseline of PHI and with low probability of spontaneous virologic control after treatment stop. Conversely, however, presence of 268E/358T does not predict control of viremia. These residues in HIV gp120 might affect in vivo HIV-1 fitness either at the level of Env function or influence susceptibility to adaptive or innate immune response.


Predicting the temporal activity patterns of new venues.
Estimating revenue and business demand of a newly opened venue is paramount as these early stages often involve critical decisions such as first rounds of staffing and resource allocation. Traditionally, this estimation has been performed through coarse-grained measures such as observing numbers in local venues or venues at similar places (e.g., coffee shops around another station in the same city). The advent of crowdsourced data from devices and services carried by individuals on a daily basis has opened up the possibility of performing better predictions of temporal visitation patterns for locations and venues. In this paper, using mobility data from Foursquare, a location-centric platform, we treat venue categories as proxies for urban activities and analyze how they become popular over time. The main contribution of this work is a prediction framework able to use characteristic temporal signatures of places together with k-nearest neighbor metrics capturing similarities among urban regions, to forecast weekly popularity dynamics of a new venue establishment in a city neighborhood. We further show how we are able to forecast the popularity of the new venue after one month following its opening by using locality and temporal similarity as features. For the evaluation of our approach we focus on London. We show that temporally similar areas of the city can be successfully used as inputs of predictions of the visit patterns of new venues, with an improvement of 41% compared to a random selection of wards as a training set for the prediction task. We apply these concepts of temporally similar areas and locality to the real-time predictions related to new venues and show that these features can effectively be used to predict the future trends of a venue. Our findings have the potential to impact the design of location-based technologies and decisions made by new business owners.


Production and characterization of polyclonal antibodies to human recombinant domain B-free antihemophilic factor VIII.
Moroctocog alpha is a human B-domain deleted recombinant factor VIII (BDDrFVIII), which represents a new generation of pure antihemophilic products. We describe here an optimized procedure for polyclonal anti-FVIII-antibody production with the use of BDDrFVIII as an immunogen. The main immunochemical characteristics of the produced antibodies and their potential biomedical applications are also reported. Rabbits were immunized with BDDrFVIII as an emulsion with Freund's adjuvant or with antigen immobilized in polyacrylamide gel (PAAG). Antibody titers in immune sera were assayed by enzyme-linked immunosorbent assay (ELISA). IgG purification was performed by afine chromatography on protein A-sepharose. Immune sera and IgG were tested by immunoblotting with the use of human plasma of healthy donors and people with hemophilia A, platelet lysates, and commercial plasma-derived concentrates as sources of FVIII-related antigens. FVIII-producing human umbilical vein cells were processed for immunocytochemical staining with the use of purified anti-FVIII-antibodies. Immunization of rabbits with PAAG-trapped antigen induced more potent immune response compared to the standard immunization procedure with Freund's adjuvant. The lowest working amount of immune IgG, measured by ELISA, was ~50 ng. Immunoblotting demonstrated that anti-BDDrFVIII antibodies effectively recognize the whole FVIII molecule (320 kDa), as well as different truncated polypeptides thereof, and are suitable for immunocytochemical analysis of FVIII-producing cells. An optimized procedure for the production of polyclonal antibodies against FVIII with the use of PAAG-immobilized BDDrFVIII (moroctocog alpha) was proposed and successfully validated. The produced antibodies are suitable for detecting and measuring FVIII-related antigens and may have various biomedical applications.


US and UK versions of the EQ-5D preference weights: does choice of preference weights make a difference?
Most US studies that estimate EQ-5D index score generally apply the UK preference weights. We compared the validity of a newly-developed US weights to the UK weights for use of EQ-5D as a measure of health-related quality of life. Data were collected from a randomized clinical trial for patients with HIV (n = 1,126) in the US. Convergent validity was examined by comparing Pearson correlations of EQ-5D index scores with the MOS-HIV Health Survey scale scores and Physical and Mental Health Summary (PHS, MHS) scores using the US and UK weights. Known-groups validity of EQ-5D US versus UK index scores was compared using clinical variables (CD4+ cell count and HIV viral load), and the MOS-HIV PHS and MHS. Score changes in the EQ-5D index from baseline to week 50 were examined using effect size (ES) estimates. The mean EQ-5D index scores was slightly higher using US weights than UK weights (0.87 vs. 0.84, respectively). The correlation coefficient for EQ-5D utilities using the US and UK weights was 0.98. The correlations of EQ-5D index scores with the MOS-HIV scores were moderate and similar using the US and UK weights. The EQ-5D index scores discriminated equally well for both versions between levels of CD4+ count, HIV viral load, and PHS and MHS scores (P < 0.05), suggesting equivalent known-groups validity. The changes in EQ-5D index scores from baseline to week 50 were similar for both versions (ES: 0.21 vs. 0.22 for US and UK, respectively), suggesting equivalent responsiveness to score changes. EQ-5D index scores generated using UK and US preference weights showed equivalent psychometric properties. For assessing treatment benefit in a single population, the use of either the UK or US weights as a measure of HRQOL will not change inferences. However, for comparisons across US and UK populations, the choice between these two weights should be based on their relevance to the study population.


Use of cracked maize as a carrier for NDV4 vaccine in experimental vaccination of chickens.
The suitability of V4 vaccine coated on cracked local grain (maize) and its husks and used for oral vaccination of chickens was assessed. Seventy-two (72) birds aged three (3) weeks and above were divided into six groups of twelve (12) birds per group. The birds were bled to determine their prevaccination HI antibody status while five different samples of cracked maize were coated with the V4 vaccine and fed to the chickens orally in each of the groups. All birds in the group including the controls were bled at 7, 14 and 21 days post vaccination to determine the presence and level of antibody response in each of the groups. Results obtained showed that prevaccination haemagglutination inhibition (HI) titre was less than two (log2) in 18% of the birds used in this experiment, however 14% of the birds had an HI titre of < or = 4. The post vaccination antibody titre showed that birds vaccinated with vaccine coated maize gave a post vaccination HI antibody titre of between Log2(6-8). when the coated maize samples were soaked in water at room temperature and assessed after 24 hours, the treated maize parts gave >6.3 log10 EID50 and above while the untreated parts gave < 3.0 log10 EID50. The experiment showed that whole maize and husks, which were not treated, may contain agents which are virus inhibitory. Form this research the treated maize which was soaked and washed gave a higher geometric mean titre, hence tends to be good carriers of the virus (vaccine). It is therefore concluded from this work that processed cracked maize could be a good carrier of NDV4 vaccine. It is hereby recommended that only treated maize could be used as carrier for the V4 vaccine.


Metabolic disposition of simvastatin in patients with T-tube drainage.
A study to investigate the disposition and biliary excretion of simvastatin (SV) was conducted in four cholecystectomy patients with T-tube drainage. Each patient received a single oral dose of 100 mg of [14C]SV (20 microCi). Of the 14C-labeled dose, approximately 35% was excreted in urine, 25% in bile, and 20% in feces. Thus, at least 60% of the oral dose was absorbed from the gastrointestinal tract. Of the AUC for radioactivity in plasma, 13% was contributed by the HMG-CoA reductase inhibitors. In addition, only 2% of the 14C-dose was eliminated in urine as HMG-CoA reductase inhibitors. Thus, most of the SV-related compounds in plasma and urine have little or no HMG-CoA reductase inhibitory activity. The same is probably true for these compounds in bile. Two major active metabolites were present in the bile. Based on HPLC and MS/MS data, they were identified as 6' beta-COOH-SVA and 6'-OH-SVA. In general, the majority of the radioactivity in the bile and urine was excreted within 24 hr postdose. Of the radioactivity excreted in the 0- to 24-hr bile, on average, approximately 30% was contributed by 6' beta-COOH-SVA and 6'-OH-SVA. These two metabolites accounted for the majority of HMG-CoA reductase inhibitory activity in the bile. Little or SV or no SVA was present in the bile.


Understanding the roles of faith-based health-care providers in Africa: review of the evidence with a focus on magnitude, reach, cost, and satisfaction.
At a time when many countries might not achieve the health targets of the Millennium Development Goals and the post-2015 agenda for sustainable development is being negotiated, the contribution of faith-based health-care providers is potentially crucial. For better partnership to be achieved and for health systems to be strengthened by the alignment of faith-based health-providers with national systems and priorities, improved information is needed at all levels. Comparisons of basic factors (such as magnitude, reach to poor people, cost to patients, modes of financing, and satisfaction of patients with the services received) within faith-based health-providers and national systems show some differences. As the first report in the Series on faith-based health care, we review a broad body of published work and introduce some empirical evidence on the role of faith-based health-care providers, with a focus on Christian faith-based health providers in sub-Saharan Africa (on which the most detailed documentation has been gathered). The restricted and diverse evidence reported supports the idea that faith-based health providers continue to play a part in health provision, especially in fragile health systems, and the subsequent reports in this Series review controversies in faith-based health care and recommendations for how public and faith sectors might collaborate more effectively.


Characterization and Genomic Analyses of Pseudomonas aeruginosa Podovirus TC6: Establishment of Genus Pa11virus.
Phages have attracted a renewed interest as alternative to chemical antibiotics. Although the number of phages is 10-fold higher than that of bacteria, the number of genomically characterized phages is far less than that of bacteria. In this study, phage TC6, a novel lytic virus of Pseudomonas aeruginosa, was isolated and characterized. TC6 consists of an icosahedral head with a diameter of approximately 54 nm and a short tail with a length of about 17 nm, which are characteristics of the family Podoviridae. TC6 can lyse 86 out of 233 clinically isolated P. aeruginosa strains, thus showing application potentials for phage therapy. The linear double-stranded genomic DNA of TC6 consisted of 49796 base pairs and was predicted to contain 71 protein-coding genes. A total of 11 TC6 structural proteins were identified by mass spectrometry. Comparative analysis revealed that the P. aeruginosa phages TC6, O4, PA11, and IME180 shared high similarity at DNA sequence and proteome levels, among which PA11 was the first phage discovered and published. Meanwhile, these phages contain 54 core genes and have very close phylogenetic relationships, which distinguish them from other known phage genera. We therefore proposed that these four phages can be classified as Pa11virus, comprising a new phage genus of Podoviridae that infects Pseudomonas spp. The results of this work promoted our understanding of phage biology, classification, and diversity.


MetroHealth Care Plus: Effects Of A Prepared Safety Net On Quality Of Care In A Medicaid Expansion Population.
Studies of Medicaid expansion have produced conflicting results about whether the expansion is having a positive impact on health and the cost and efficiency of care delivery. To explore the issue further, we examined MetroHealth Care Plus, a Centers for Medicare and Medicaid Services (CMS) waiver program in Ohio composed of three safety-net organizations that enrolled 28,295 uninsured poor patients in closed-panel care during 2013. All participating organizations used electronic health records and patient-centered medical homes, publicly reported performance in a regional health improvement collaborative, and accepted a budget-neutral cap approved by CMS. We compared changes between 2012 and 2013 in achieving quality standards for diabetes and hypertension among 3,437 MetroHealth Care Plus enrollees to changes among 1,150 patients with the same conditions who remained uninsured in both years. Compared to continuously uninsured patients with diabetes, MetroHealth Care Plus enrollees with diabetes improved significantly more on composite standards of care and intermediate outcomes. Among enrollees with hypertension, blood pressure control improvements were insignificantly larger than those in the continuously uninsured group with hypertension. Across all 28,295 enrollees, 2013 total costs of care were 28.7 percent below the budget cap, providing cause for optimism that a prepared safety net can meet the challenges of Medicaid expansion.


Regiospecific gamma-conjugation of methotrexate to poly(L-lysine). Chemical and biological studies.
Regiospecific syntheses of gamma- and alpha-conjugates of methotrexate and poly(L-lysine) are described. The alpha- and gamma-t-butyl esters, respectively, of methotrexate were coupled to poly(L-lysine) with diphenylphosphoryl azide in N,N-dimethylformamide, the ester-protecting group was cleaved with 15% hydrogen bromide in acetic acid, and small molecules were removed by dialysis. Poly(L-lysine) of Mr = 1,500-8,000 and 8,000-30,000 was used to prepare six different conjugates, which were characterized by ultraviolet absorbance measurement and quantitative amino acid analysis. The degree of substitution varied from one methotrexate per 4.7 lysines to one methotrexate per 10.2 lysines. Dihydrofolate reductase inhibition in a cell-free assay was observed with alpha- and gamma-conjugates, but the latter had the greater affinity (only 3-fold less than that of methotrexate itself). The binding of the conjugates exhibited a slight pH dependence, with affinity being greater at pH 7.2 than at pH 8.5 for both alpha- and gamma-conjugates. Toxicity to cultured rat hepatoma cells (H35) was also greater for the gamma-conjugates, and showed some dependence on the chain-length and degree of substitution of the poly(L-lysine) carrier. Cells resistant to methotrexate by virtue of a transport defect (H35R0.3 line) retained their sensitivity to the gamma-conjugate, but less so to the alpha-conjugate. There was also some retention of sensitivity in a more highly resistant cell line (H35R10) with impaired methotrexate transport and a concomitant increase in dihydrofolate reductase activity. gamma-Conjugation was likewise more favorable in cytotoxicity assays against L1210 murine leukemia cells, and there was partial retention of activity against highly methotrexate-resistant lines (L1210/R71 and L1210/R81) with a transport defect and/or an elevation of dihydrofolate reductase content. In antitumor assays against intraperitoneal L1210 leukemia in mice, a gamma-conjugate with Mr = 8,000-30,000 and one methotrexate per 5.5 lysines produced a 35-75% increase in lifespan when administered intraperitoneally at single doses equivalent to 10-20 mg/kg of methotrexate. A similar increase in lifespan with methotrexate alone on the single-dose regimen required 50-150 mg/kg. An alpha-conjugate of similar Mr and degree of substitution was inactive at nontoxic doses, as were other gamma-conjugates of lower Mr and/or degree of substitution.(ABSTRACT TRUNCATED AT 400 WORDS)


VASP-E: specificity annotation with a volumetric analysis of electrostatic isopotentials.
Algorithms for comparing protein structure are frequently used for function annotation. By searching for subtle similarities among very different proteins, these algorithms can identify remote homologs with similar biological functions. In contrast, few comparison algorithms focus on specificity annotation, where the identification of subtle differences among very similar proteins can assist in finding small structural variations that create differences in binding specificity. Few specificity annotation methods consider electrostatic fields, which play a critical role in molecular recognition. To fill this gap, this paper describes VASP-E (Volumetric Analysis of Surface Properties with Electrostatics), a novel volumetric comparison tool based on the electrostatic comparison of protein-ligand and protein-protein binding sites. VASP-E exploits the central observation that three dimensional solids can be used to fully represent and compare both electrostatic isopotentials and molecular surfaces. With this integrated representation, VASP-E is able to dissect the electrostatic environments of protein-ligand and protein-protein binding interfaces, identifying individual amino acids that have an electrostatic influence on binding specificity. VASP-E was used to examine a nonredundant subset of the serine and cysteine proteases as well as the barnase-barstar and Rap1a-raf complexes. Based on amino acids established by various experimental studies to have an electrostatic influence on binding specificity, VASP-E identified electrostatically influential amino acids with 100% precision and 83.3% recall. We also show that VASP-E can accurately classify closely related ligand binding cavities into groups with different binding preferences. These results suggest that VASP-E should prove a useful tool for the characterization of specific binding and the engineering of binding preferences in proteins.


Development of a gill perfusion apparatus for studying the interaction of fish pathogens with gill tissue.
An isolated perfused gill preparation was developed for the study of the association of gill pathogens with the branchial tissue. The preparation consisted of an excised branchial arch from common carp (Cyprinus carpio L., minimum weight 300g), perfused via the afferent branchial artery. Filtered and heparinised Cortland solution was used as the perfusion fluid and infused by means of a drip (3-litre bag). The average perfusion rate was 1.5ml/minute/arch/kg body weight. The outflowing perfusate was collected from a cannula in the efferent branchial artery. The individual gill arch was suspended in a circular organ chamber filled with Ringer solution, which was aerated and kept at a constant temperature of 20°C. Unperfused gill arches maintained in Ringer solution at the same temperature served as controls. Cortland solution proved to be a satisfactory perfusion fluid, maintaining the perfused gills in a healthy condition for at least 4 hours with no, or only slight, oedema after 90 minutes, and slight or moderate oedema after 4 hours. The unperfused gill displayed excessive necrosis and loss of architecture after 4 hours. The Cortland perfused gill apparatus could thus prove to be an alternative ex vivo model of particular use in the study of the early interaction of gill associated pathogens with the branchial tissue.


Serum leptin measured in early pregnancy is higher in women with preeclampsia compared with normotensive pregnant women.
Leptin, an adipocyte-derived hormone, plays an important role in reproduction and angiogenesis. Studies examining leptin in preeclampsia are inconsistent, possibly because of small sample sizes and variability in sampling and outcome. We conducted a nested case-control study to examine associations between serum leptin (measured: 9-26 weeks gestation) and preeclampsia among 430 primiparous preeclamptic women and 316 primiparous normotensive controls from the Danish National Birth Cohort. Median (interquartile range) leptin concentrations were calculated. Associations between leptin and preeclampsia (blood pressure ≥140/90 mm Hg), term preeclampsia (preeclampsia and delivery ≥37 weeks gestation), or preterm preeclampsia (preeclampsia and delivery <37 weeks gestation) were examined using generalized linear models adjusting for body mass index, gestational age at blood draw, maternal age, smoking, and socio-occupational status. As leptin is increased in obese women and the risk of preeclampsia increases with body mass index, we used the Sobel test to examine whether leptin is a mediator of this relationship. After adjustments, leptin concentrations were significantly higher in women with preeclampsia (30.5 [24.6]; P=0.0117) and term preeclampsia (30.4 [24.9]; P=0.0228) compared with controls (20.9 [28.3]). There was no significant difference between preterm preeclampsia (30.6 [23.4]; P=0.2210) and controls. Leptin is a possible mediator of the association between body mass index and preeclampsia (P=0.0276). Leptin concentrations are higher in women with preeclampsia compared with normotensive controls and may mediate some of the relationship between body mass index and preeclampsia.


The influence of preoperative total parenteral nutrition upon morbidity and mortality.
This study was done to examine the criteria for determining the adequacy of preoperative total parenteral nutrition (TPN). We hypothesized that an important criterion for adequate preoperative nutrition should be a contraction of the expanded extracellular fluid compartment known to occur in malnourished patients undergoing surgical treatment. Clinical evidence of this included a rise in the serum albumin level and weight loss. Fifty-nine patients requiring preoperative nutritional support for at least five days were admitted to the study. The patients were divided into three groups (group 1, group 2a and group 2b) based upon the response of the patients to TPN. Group 1 consisted of 23 patients who demonstrated a rise in serum albumin value, loss in body weight and diuresis after seven days of TPN. Group 2 was made up of 36 patients who failed to demonstrate a rise in the albumin level in response to nutritional support for one week. Of these patients, 20 underwent operation at the end of a week of nutritional support (group 2a) while 16 patients received four to six weeks of nutritional support preoperatively (group 2b). The complication rate in the three groups was 4.3 per cent for those in group 1; 45 per cent for those in group 2a and 12.5 per cent for those in group 2b (p less than 0.05), group 2a versus group 2b). The results of this study demonstrate a high mortality and morbidity in patients who fail to increase the serum albumin level after one week of TPN. The data suggest that a prolonged period of parenteral nutrition results in a substantial decrease in perioperative complications in this group of patients.


Cognition, mood disorders, and sex hormones.
Macaques (Macaca spp.) are useful models to evaluate effects of ovarian sex steroids and selective estrogen receptor modulators (SERMs) on mood and cognitive function due to similarities to women in their reproductive and central nervous systems. The results of nonhuman primate studies support the hypothesis that estrogen mediates specific aspects of attention and memory, yet much work is needed to understand which cognitive processes are affected, whether natural versus surgical menopause effects are different, and the interaction of age and ovarian senescence on cognitive function. This knowledge is necessary to determine whether to support the cognitive function of women in the menopausal phase of life and, if so, to determine efficacious therapeutic interventions. Mood disorders are prevalent in women and are associated with reproductive function in women and macaques. Exogenous steroid therapies, including oral contraceptives and postmenopausal hormone replacement therapies, have behavioral effects in women and appear to affect the behavior and underlying neural substrates of monkeys. Additional research is necessary to confirm and extend these observations. Ovarian steroids have multiple effects on serotonin synthesis, reuptake, and degradation, on neural activity that drives serotonin release, and on receptor activation in primates. This system modulates cognitive function and mood and is the target of a broad class of antidepressant therapies. Understanding the effects of ovarian steroids on the neural serotonergic system is necessary to understand depression in women. These studies are best carried out in primate models, which are more similar to humans in neural serotonergic function than other animal models.


A catalogue of validated single nucleotide polymorphisms in bovine orthologs of mammalian imprinted genes and associations with beef production traits.
Genetic (or 'genomic') imprinting, a feature of approximately 100 mammalian genes, results in monoallelic expression from one of the two parentally inherited chromosomes. To date, most studies have been directed on imprinted genes in murine or human models; however, there is burgeoning interest in the effects of imprinted genes in domestic livestock species. In particular, attention has focused on imprinted genes that influence foetal growth and development and that are associated with several economically important production traits in cattle, sheep and pigs. We have re-sequenced regions in 20 candidate bovine imprinted genes in order to validate single nucleotide polymorphisms (SNPs) that may influence important production traits in cattle. Putative SNPs detected via re-sequencing were subsequently re-formatted for high-throughput SNP genotyping in 185 cattle samples comprising 138 performance-tested European Bos taurus (all Limousin bulls), 29 African B. taurus and 18 Indian B. indicus samples. Analysis of the resulting genotypic data identified 117 validated SNPs. Preliminary genotype-phenotype association analyses using 83 SNPs that were polymorphic in the Limousin samples with minor allele frequencies ⩾0.05 revealed significant associations between two candidate bovine imprinted genes and a range of important beef production traits: average daily gain, average feed intake, live weight, feed conversion ratio, residual feed intake and residual gain. These genes were the Ras protein-specific guanine nucleotide releasing factor gene (RASGRF1) and the zinc finger, imprinted 2 gene (ZIM2). Despite the relatively small sample size used in these analyses, the observed associations with production traits are supported by the purported biological function of the RASGRF1 and ZIM2 gene products. These results support the hypothesis that imprinted genes contribute significantly to important complex production traits in cattle. Furthermore, these SNPs may be usefully incorporated into future marker-assisted and genomic selection breeding schemes.


Comparing in vivo pump-probe and multiphoton fluorescence microscopy of melanoma and pigmented lesions.
We demonstrate a multimodal approach that combines a pump-probe with confocal reflectance and multiphoton autofluorescence microscopy. Pump-probe microscopy has been proven to be of great value in analyzing thin tissue sections of pigmented lesions, as it produces molecular contrast which is inaccessible by other means. However, the higher optical intensity required to overcome scattering in thick tissue leads to higher-order nonlinearities in the optical response of melanin (e.g., two-photon pump and one-photon probe) that present additional challenges for interpreting the data. We show that analysis of pigment composition in vivo must carefully account for signal terms that are nonlinear with respect to the pump and probe intensities. We find that pump-probe imaging gives useful contrast for pigmented structures over a large range of spatial scales (100 μm to 1 cm), making it a potentially useful tool for tracking the progression of pigmented lesions without the need to introduce exogenous contrast agents.


Theoretical and experimental analysis of the structural pattern responsible for the iridescence of Morpho butterflies.
Morpho butterflies are well-known for their iridescence originating from nanostructures in the scales of their wings. These optical active structures integrate three design principles leading to the wide angle reflection: alternating lamellae layers, "Christmas tree" like shape, and offsets between neighboring ridges. We study their individual effects rigorously by 2D FEM simulations of the nanostructures of the Morpho sulkowskyi butterfly and show how the reflection spectrum can be controlled by the design of the nanostructures. The width of the spectrum is broad (≈ 90 nm) for alternating lamellae layers (or "brunches") of the structure while the "Christmas tree" pattern together with a height offset between neighboring ridges reduces the directionality of the reflectance. Furthermore, we fabricated the simulated structures by e-beam lithography. The resulting samples mimicked all important optical features of the original Morpho butterfly scales and feature the intense blue iridescence with a wide angular range of reflection.


Cross-Cultural Adaptation and Validation of the Voice Handicap Index in the Quebec French Population (VHI-QF).
The Voice Handicap Index (VHI) is a widely recognized, self-administered questionnaire, designed to evaluate patients' perception of voice-related disability. It takes into consideration the physical, functional and emotional impacts of dysphonia. The VHI has been translated and validated in many languages, including European French. The purpose of our study is to translate, adapt and validate a new version of the VHI in Quebec French. The original VHI was translated into Quebec French (QF) by forward and backward translations by four professional translators, including a speech-language pathologist. The content validity of the resulting VHI-QF was examined in focus groups with six patients and seven speech-language pathologists. Another sample of 154 patients with voice disorders and 150 healthy controls allowed evaluation of the new questionnaire's convergent and discriminant validity, and internal consistency. Satisfaction toward the questionnaire was also evaluated for all patients, as well Test-retest reliability and responsiveness for a sub-sample. The VHI-QF showed a moderate correlation with dysphonia severity level, indicating adequate convergent validity. Both total and subscale scores also exhibited adequate ability to discriminate between patients and controls (discriminant validity), high internal consistency, and good test-retest reliability. The analysis of pre- and post-treatment VHI-QF scores revealed adequate responsiveness to voice treatment. Patients were overall satisfied with the questionnaire. The VHI-QF is a valid, reliable and clinically useful self-reported tool to evaluate the severity and change of voice disorders in Quebec French population. Therefore this questionnaire can be used in clinical and research contexts.


Development of a Novel Adult Congenital Heart Disease-Specific Patient-Reported Outcome Metric.
Background Patient-reported outcome metrics (PROs) quantify important outcomes in clinical trials and can be sensitive measures of patient experience in clinical practice. Currently, there is no validated disease-specific PRO for adults with congenital heart disease (ACHD). Methods and Results We conducted a preliminary psychometric validation of a novel ACHD PRO. ACHD patients were recruited prospectively from 2 institutions and completed a series of questionnaires, a physician health assessment, and a 6-minute walk test. Participants returned to complete the same questionnaires and assessment 3 months±2 weeks later. We tested the internal consistency and test-retest reliability by comparing responses among clinically stable patients at the 2 study visits. We assessed convergent and divergent validity by comparison of ACHD PRO responses to existing validated questionnaires. We assessed responsiveness by comparison with patient-reported clinical change. One hundred three patients completed 1 study visit and 81 completed both. The ACHD PRO demonstrated good internal consistency in each of its 5 domains (Cronbach's α: 0.87; 0.74; 0.74; 0.90; and 0.89, respectively) and in the overall summary score (0.92). Test-retest reliability was good with an intraclass correlation ≥0.73 for all domains and 0.78 for the Summary Score. The ACHD PRO accurately assessed domain concepts based on comparison with validated standards. Preliminary estimates of responsiveness suggest sensitivity to clinical status. Conclusions These studies provide initial support for the validity and reliability of the ACHD PRO. Further studies are needed to assess its sensitivity to changes in clinical status.


Mainstreaming Underutilized Indigenous and Traditional Crops into Food Systems: A South African Perspective.
Business as usual or transformative change? While the global agro-industrial food system is credited with increasing food production, availability and accessibility, it is also credited with giving birth to 'new' challenges such as malnutrition, biodiversity loss, and environmental degradation. We reviewed the potential of underutilized indigenous and traditional crops to bring about a transformative change to South Africa's food system. South Africa has a dichotomous food system, characterized by a distinct, dominant agro-industrial, and, alternative, informal food system. This dichotomous food system has inadvertently undermined the development of smallholder producers. While the dominant agro-industrial food system has led to improvements in food supply, it has also resulted in significant trade-offs with agro-biodiversity, dietary diversity, environmental sustainability, and socio-economic stability, especially amongst the rural poor. This challenges South Africa's ability to deliver on sustainable and healthy food systems under environmental change. The review proposes a transdisciplinary approach to mainstreaming underutilized indigenous and traditional crops into the food system, which offers real opportunities for developing a sustainable and healthy food system, while, at the same time, achieving societal goals such as employment creation, wellbeing, and environmental sustainability. This process can be initiated by researchers translating existing evidence for informing policy-makers. Similarly, policy-makers need to acknowledge the divergence in the existing policies, and bring about policy convergence in pursuit of a food system which includes smallholder famers, and where underutilized indigenous and traditional crops are mainstreamed into the South African food system.


Drug-eluting stents show delayed healing: paclitaxel more pronounced than sirolimus.
To understand wound healing after drug-eluting stents (DES) placement in humans, we studied the histology of in-stent restenosis (ISR) tissue obtained by atherectomy from bare metal stents (BMS) and DES in comparison with de novo atherosclerosis. The tissue was retrieved from ISR in ten sirolimus-eluting stents (SES) and nine paclitaxel-eluting stents (PES), six BMS, and nine stenotic de novo atherosclerotic lesions and processed for histology and immunocytochemistry. Patients with ISR in PES showed a significantly higher incidence of unstable angina upon presentation for re-intervention (P = 0.046). De novo tissue tended to be more collagen rich, whereas ISR tissue tended to be more proteoglycan rich. In all groups, cell content consisted almost exclusively of smooth muscle cells. Histology showed that fibrinoid in ISR tissue was present only in DES (P = 0.004), as late as 2 years following DES placement, indicating a persistent incomplete healing response. The amount of fibrinoid, given as a percentage of total tissue in each atherectomy specimen, was greater in PES than in SES (17 vs. 5%, P = 0.026). ISR in DES shows incomplete neointimal healing as late as 2 years after implantation. Patients with ISR in PES presented with more unstable angina and showed more pronounced signs of delayed healing than SES.


Cytotoxic, Antiproliferative and Pro-Apoptotic Effects of 5-Hydroxyl-6,7,3',4',5'-Pentamethoxyflavone Isolated from Lantana ukambensis.
Lantana ukambensis (Vatke) Verdc. is an African food and medicinal plant. Its red fruits are eaten and highly appreciated by the rural population. This plant was extensively used in African folk medicinal traditions to treat chronic wounds but also as anti-leishmanial or cytotoxic remedies, especially in Burkina Faso, Tanzania, Kenya, or Ethiopia. This study investigates the in vitro bioactivity of polymethoxyflavones extracted from a L. ukambensis as anti-proliferative and pro-apoptotic agents. We isolated two known polymethoxyflavones, 5,6,7,3',4',5'-hexamethoxyflavone (1) and 5-hydroxy-6,7,3',4',5'-pentamethoxyflavone (2) from the whole plant of L. ukambensis. Their chemical structures were determined by spectroscopic analysis and comparison with published data. These molecules were tested for the anti-proliferative, cytotoxic and pro-apoptotic effects on human cancer cells. Among them, 5-hydroxy-6,7,3',4',5'-pentamethoxyflavone (2) was selectively cytotoxic against monocytic lymphoma (U937), acute T cell leukemia (Jurkat), and chronic myelogenous leukemia (K562) cell lines, but not against peripheral blood mononuclear cells (PBMCs) from healthy donors, at all tested concentrations. Moreover, this compound exhibited significant anti-proliferative and pro-apoptotic effects against U937 acute myelogenous leukemia cells. This study highlights the anti-proliferative and pro-apoptotic effects of 5-hydroxy-6,7,3',4',5'-pentamethoxyflavone (2) and provides a scientific basis of traditional use of L. ukambensis.


Activation of Myeloid TLR4 Mediates T Lymphocyte Polarization after Traumatic Brain Injury.
Traumatic brain injury (TBI) is a major public health issue, producing significant patient mortality and poor long-term outcomes. Increasing evidence suggests an important, yet poorly defined, role for the immune system in the development of secondary neurologic injury over the days and weeks following a TBI. In this study, we tested the hypothesis that peripheral macrophage infiltration initiates long-lasting adaptive immune responses after TBI. Using a murine controlled cortical impact model, we used adoptive transfer, transgenic, and bone marrow chimera approaches to show increased infiltration and proinflammatory (classically activated [M1]) polarization of macrophages for up to 3 wk post-TBI. Monocytes purified from the injured brain stimulated the proliferation of naive T lymphocytes, enhanced the polarization of T effector cells (TH1/TH17), and decreased the production of regulatory T cells in an MLR. Similarly, elevated T effector cell polarization within blood and brain tissue was attenuated by myeloid cell depletion after TBI. Functionally, C3H/HeJ (TLR4 mutant) mice reversed M1 macrophage and TH1/TH17 polarization after TBI compared with C3H/OuJ (wild-type) mice. Moreover, brain monocytes isolated from C3H/HeJ mice were less potent stimulators of T lymphocyte proliferation and TH1/TH17 polarization compared with C3H/OuJ monocytes. Taken together, our data implicate TLR4-dependent, M1 macrophage trafficking/polarization into the CNS as a key mechanistic link between acute TBI and long-term, adaptive immune responses.


A high-throughput multi-microfluidic crystal generator (MMicroCryGen) platform for facile screening of polymorphism and crystal morphology for pharmaceutical compounds.
In this work, a novel multi-microfluidic crystallization platform called MMicroCryGen is presented, offering a facile methodology for generating individual crystals for fast and easy screening of the polymorphism and crystal habit of solid compounds. The MMicroCryGen device is capable of performing 8 × 10 cooling crystallization experiments in parallel using 8 disposable microcapillary film strips, each requiring less than 25 μL of solution. Compared to traditional microfluidic systems, the MMicroCryGen platform does not require complex fluid handling; it can be directly integrated with a 96-well microplate and it can also work in a "dipstick" mode. The produced crystals can be safely and directly observed inside the capillaries by optical and spectroscopic techniques. The platform was validated by performing a number of independent experimental runs for: (1) polymorph and hydrate screening of ortho-aminobenzoic acid, succinic acid and piroxicam; (2) co-crystal form screening of the p-toluenesulfonamide/triphenylphosphine oxide system; (3) studying the effect of o-toluic acid on ortho-aminobenzoic cooling crystallization (effect of structurally related additives). In all three cases, all known solid forms were identified with a single experiment using ∼200 μL of solvent and just a few micrograms of the solid material. The MMicroCryGen is simple to use, inexpensive and it provides increased flexibility compared to traditional crystallization techniques, being an effective new microfluidic solution for solid form screening in pharmaceutical, fine chemicals, food and agrochemical industries.


Lmna knockout mouse embryonic fibroblasts are less contractile than their wild-type counterparts.
In order to maintain tissue homeostasis and functionality, adherent cells need to sense and respond to environmental mechanical stimuli. An important ability that adherent cells need in order to properly sense and respond to mechanical stimuli is the ability to exert contractile stress onto the environment via actin stress fibers. The actin stress fibers form a structural chain between the cells' environment via focal adhesions and the nucleus via the nuclear lamina. In case one of the links in this chain is missing or aberrant, contractile stress generation will be affected. This is especially the case in laminopathic cells, which have a missing or mutated form of the LMNA gene encoding for part of the nuclear lamina. Using the thin film method combined with sample specific finite element modeling, we quantitatively showed a fivefold lower contractile stress generation of Lmna knockout mouse embryonic fibroblasts (MEFs) as compared to wild-type MEFs. Via fluorescence microscopy it was demonstrated that the lower contractile stress generation was associated with an impaired actin stress fiber organization with thinner actin fibers and smaller focal adhesions. Similar experiments with wild-type MEFs with chemically disrupted actin stress fibers verified these findings. These data illustrate the importance of an organized actin stress fiber network for contractile stress generation and demonstrate the devastating effect of an impaired stress fiber organization in laminopathic fibroblasts. Next to this, the thin film method is expected to be a promising tool in unraveling contractility differences between fibroblasts with different types of laminopathic mutations.


Mediterranean lifestyle and cardiovascular disease prevention.
Adherence to a Mediterranean dietary pattern is a well-established protective factor against cardiovascular disease (CVD). However, diet quality is only one aspect of the overall healthy lifestyle adopted by Mediterranean populations. The latter has never been evaluated as a multi-factorial composite lifestyle. Thus, the aim of the present study was to provide a broader picture of the Mediterranean lifestyle and its effects on CVD risk, among elderly individuals. During 2005-2015, 2,749 older (aged 65-100 years) from 21 Mediterranean islands (MEDIS) and the rural Mani region (Peloponnesus) of Greece were voluntarily enrolled onto the study. Dietary habits, physical activity status, socio-demographic characteristics, lifestyle parameters (sleep, smoking habits, social life and educational status) and clinical profile aspects were derived through standard procedures. The overall prevalence of the traditional CVD risk factors were 62.3% for hypertension, 22.3% for diabetes mellitus (type 2) and 47.7% for hypercholesterolemia. The presence of diabetes mellitus was positively predicted by the geriatric depression scale (GDS) [odds ratio (OR) =1.13, 95% confidence interval (CI): 1.02-1.25] and by an urban residential environment (OR =2.57, 95% CI: 1.10-6.06) after adjusting for several confounders. Presence of hypertension was predicted by increasing age (OR =1.07, 95% CI: 1.02-1.12), increasing body mass index (BMI) (OR =1.12, 95% CI: 1.04-1.21), the habit of midday sleep (OR =2.07, 95% CI: 1.07-4.02) and inversely predicted by the frequency of socializing with friends (OR =0.767, 95% CI: 0.616-0.955). The estimated score in the GDS was the only independent positive predictor for the presence of hypercholesterolemia (OR =1.10, 95% CI: 1.01-1.21). Lifestyle parameters such as social life, midday sleep (siesta) and residential environment are strongly associated with the presence of CVD risk factors in elderly and should be part of broader CVD prevention strategies to reduce the burden of the disease.


Is being breastfed as an infant associated with adult pulmonary function?
Breastfeeding reduces the risk of asthma and respiratory infections in infants. Since respiratory infections are associated with reduced pulmonary function in adolescents, pulmonary function impairment may be carried into adulthood. Our aim was to determine whether a history of having been breastfed as an infant is a determinant of adult pulmonary function. We analyzed data from a general population sample of residents of Erie and Niagara Counties between September 1995 and December 1999. We calculated forced expiratory volume in one second (FEV(1)) and forced vital capacity (FVC) prediction equations and used multiple linear regression models to study the association between having been breastfed as an infant and percentage predicted FEV(1) (FEV(1)%) and percentage predicted FVC (FVC%) after adjustment for covariates. Of 2305 subjects, 62% reported having been breastfed. After controlling for age, gender, weight, smoking status, pack-years of smoking, eosinophil counts and dietary factors, there was no association between having been breastfed (yes/no) and FEV(1)% or FVC% (regression coefficients 0.0049, p = 0.46 and 0.0055, p = 0.43, respectively). We did not find a strong or consistent association between having been breastfed as an infant and pulmonary function in adulthood.


Levels and predictors of exercise referral scheme uptake and adherence: a systematic review.
The effectiveness of exercise referral schemes (ERS) is influenced by uptake and adherence to the scheme. The identification of factors influencing low uptake and adherence could lead to the refinement of schemes to optimise investment. To quantify the levels of ERS uptake and adherence and to identify factors predictive of uptake and adherence. A systematic review and meta-analysis was undertaken. MEDLINE, EMBASE, PsycINFO, Cochrane Library, ISI WOS, SPORTDiscus and ongoing trial registries were searched (to October 2009) and included study references were checked. Included studies were required to report at least one of the following: (1) a numerical measure of ERS uptake or adherence and (2) an estimate of the statistical association between participant demographic or psychosocial factors (eg, level of motivation, self-efficacy) or programme factors and uptake or adherence to ERS. Twenty studies met the inclusion criteria, six randomised controlled trials (RCTs) and 14 observational studies. The pooled level of uptake in ERS was 66% (95% CI 57% to 75%) across the observational studies and 81% (95% CI 68% to 94%) across the RCTs. The pooled level of ERS adherence was 49% (95% CI 40% to 59%) across the observational studies and 43% (95% CI 32% to 54%) across the RCTs. Few studies considered anything other than gender and age. Women were more likely to begin an ERS but were less likely to adhere to it than men. Older people were more likely to begin and adhere to an ERS. Substantial heterogeneity was evident across the ERS studies. Without standardised definitions, the heterogeneity may have been reflective of differences in methods of defining uptake and adherence across studies. To enhance our understanding of the variation in uptake and adherence across ERS and how these variations might affect physical activity outcomes, future trials need to use quantitative and qualitative methods.


Combining short-term metformin treatment and one bout of exercise does not increase insulin action in insulin-resistant individuals.
Results from the Diabetes Prevention Program highlight the effectiveness of metformin or regular physical activity in the prevention of type 2 diabetes. Independently, metformin and exercise increase insulin sensitivity, but they have not been studied in combination. To assess the combined effects, insulin-resistant subjects (n = 9) matched for weight, body fat, and aerobic fitness were studied before any treatment (B), after 2-3 wk of 2,000 mg/day metformin (MET), and after metformin plus 40 min of exercise at 65% Vo(2peak) (MET + Ex). A second group (n = 7) was studied at baseline and after an identical bout of exercise with no metformin (Ex). Biopsies of the vastus lateralis were taken at B, after MET, immediately after MET + Ex (group 1), or immediately after Ex (group 2). Insulin sensitivity was assessed 4 h postexercise with a euglycemic hyperinsulinemic (40 mU.m(2).min(-1)) clamp enriched with [6,6-(2)H]glucose. Insulin sensitivity was 54% higher after Ex (P < 0.01), but there was no change with Met + Ex. Skeletal muscle AMPKalpha2 activity was elevated threefold (P < 0.01) after Ex, but there was no increase with MET + Ex. These findings suggest that the combination of short-term metformin treatment and an acute bout of exercise does not enhance insulin sensitivity, and the addition of metformin may attenuate the well-documented effects of exercise alone.


Investigation of chromosome 1q reveals differential expression of members of the S100 family in clinical subgroups of intracranial paediatric ependymoma.
Gain of 1q is one of the most common alterations in cancer and has been associated with adverse clinical behaviour in ependymoma. The aim of this study was to investigate this region to gain insight into the role of 1q genes in intracranial paediatric ependymoma. To address this issue we generated profiles of eleven ependymoma, including two relapse pairs and seven primary tumours, using comparative genome hybridisation and serial analysis of gene expression. Analysis of 656 SAGE tags mapping to 1q identified CHI3L1 and S100A10 as the most upregulated genes in the relapse pair with de novo 1q gain upon recurrence. Moreover, three more members of the S100 family had distinct gene expression profiles in ependymoma. Candidates (CHI3L1, S100A10, S100A4, S100A6 and S100A2) were validated using immunohistochemistry on a tissue microarray of 74 paediatric ependymoma. In necrotic cases, CHI3L1 demonstrated a distinct staining pattern in tumour cells adjacent to the areas of necrosis. S100A6 significantly correlated with supratentorial tumours (P<0.001) and S100A4 with patients under the age of 3 years at diagnosis (P=0.038). In conclusion, this study provides evidence that S100A6 and S100A4 are differentially expressed in clinically relevant subgroups, and also demonstrates a link between CHI3L1 protein expression and necrosis in intracranial paediatric ependymoma.


A high quality assembly of the Nile Tilapia (Oreochromis niloticus) genome reveals the structure of two sex determination regions.
Tilapias are the second most farmed fishes in the world and a sustainable source of food. Like many other fish, tilapias are sexually dimorphic and sex is a commercially important trait in these fish. In this study, we developed a significantly improved assembly of the tilapia genome using the latest genome sequencing methods and show how it improves the characterization of two sex determination regions in two tilapia species. A homozygous clonal XX female Nile tilapia (Oreochromis niloticus) was sequenced to 44X coverage using Pacific Biosciences (PacBio) SMRT sequencing. Dozens of candidate de novo assemblies were generated and an optimal assembly (contig NG50 of 3.3Mbp) was selected using principal component analysis of likelihood scores calculated from several paired-end sequencing libraries. Comparison of the new assembly to the previous O. niloticus genome assembly reveals that recently duplicated portions of the genome are now well represented. The overall number of genes in the new assembly increased by 27.3%, including a 67% increase in pseudogenes. The new tilapia genome assembly correctly represents two recent vasa gene duplication events that have been verified with BAC sequencing. At total of 146Mbp of additional transposable element sequence are now assembled, a large proportion of which are recent insertions. Large centromeric satellite repeats are assembled and annotated in cichlid fish for the first time. Finally, the new assembly identifies the long-range structure of both a ~9Mbp XY sex determination region on LG1 in O. niloticus, and a ~50Mbp WZ sex determination region on LG3 in the related species O. aureus. This study highlights the use of long read sequencing to correctly assemble recent duplications and to characterize repeat-filled regions of the genome. The study serves as an example of the need for high quality genome assemblies and provides a framework for identifying sex determining genes in tilapia and related fish species.


Consultative (expert) second opinions in soft tissue pathology. Analysis of problem-prone diagnostic situations.
We reviewed 500 consecutive soft tissue lesions referred for expert consultation to determine types of lesions and/or situations in which major discrepancies occur. Of 266 cases (53.2%) accompanied by a diagnosis, essential agreement with the second opinion was noted in 68%, minor discrepancy in 7%, and major discrepancy in 25%. The 65 major discrepancies were distributed proportionally to the referring sources and could be divided into 4 groups: benign mesenchymal lesions diagnosed as sarcomas (45%), sarcomas diagnosed as benign tumors (23%), nonmesenchymal lesions diagnosed as sarcoma (20%), and major grading discrepancies (12%). Relatively few lesions accounted for a major proportion of major discrepancies. Problematic lesions were lipoma and fasciitis and their variants and desmoplastic-neurotropic melanoma. Needle biopsy specimens were somewhat more likely to be associated with a discrepant opinion. With the exception of nonmesenchymal lesions, the diagnosis for all major discrepant cases could be made on the basis of the H&E-stained slides, suggesting that failure to perform immunostains did not account for discrepancies. Lack of familiarity with rare or unusual lesions is probably more significant in explaining diagnostic discrepancies than is the increasing use of needle biopsy or the failure to perform immunohistochemical analysis.


Mutational signatures of de-differentiation in functional non-coding regions of melanoma genomes.
Much emphasis has been placed on the identification, functional characterization, and therapeutic potential of somatic variants in tumor genomes. However, the majority of somatic variants lie outside coding regions and their role in cancer progression remains to be determined. In order to establish a system to test the functional importance of non-coding somatic variants in cancer, we created a low-passage cell culture of a metastatic melanoma tumor sample. As a foundation for interpreting functional assays, we performed whole-genome sequencing and analysis of this cell culture, the metastatic tumor from which it was derived, and the patient-matched normal genomes. When comparing somatic mutations identified in the cell culture and tissue genomes, we observe concordance at the majority of single nucleotide variants, whereas copy number changes are more variable. To understand the functional impact of non-coding somatic variation, we leveraged functional data generated by the ENCODE Project Consortium. We analyzed regulatory regions derived from multiple different cell types and found that melanocyte-specific regions are among the most depleted for somatic mutation accumulation. Significant depletion in other cell types suggests the metastatic melanoma cells de-differentiated to a more basal regulatory state. Experimental identification of genome-wide regulatory sites in two different melanoma samples supports this observation. Together, these results show that mutation accumulation in metastatic melanoma is nonrandom across the genome and that a de-differentiated regulatory architecture is common among different samples. Our findings enable identification of the underlying genetic components of melanoma and define the differences between a tissue-derived tumor sample and the cell culture created from it. Such information helps establish a broader mechanistic understanding of the linkage between non-coding genomic variations and the cellular evolution of cancer.


Soft dentin results in unique flexible teeth in scraping catfishes.
Teeth are generally used for actions in which they experience mainly compressive forces acting toward the base. The ordered tooth enamel(oid) and dentin structures contribute to the high compressive strength but also to the minor shear and tensile strengths. Some vertebrates, however, use their teeth for scraping, with teeth experiencing forces directed mostly normal to their long axis. Some scraping suckermouth catfishes (Loricariidae) even appear to have flexible teeth, which have not been found in any other vertebrate taxon. Considering the mineralized nature of tooth tissues, the notion of flexible teeth seems paradoxical. We studied teeth of five species, testing and measuring tooth flexibility, and investigating tooth (micro)structure using transmission electron microscopy, staining, computed tomography scanning, and scanning electron microscopy-energy-dispersive spectrometry. We quantified the extreme bending capacity of single teeth (up to 180°) and show that reorganizations of the tooth (micro)structure and extreme hypomineralization of the dentin are adaptations preventing breaking by allowing flexibility. Tooth shape and internal structure appear to be optimized for bending in one direction, which is expected to occur frequently when feeding (scraping) under natural conditions. Not all loricariid catfishes possess flexible teeth, with the trait potentially having evolved more than once. Flexible teeth surely rank among the most extreme evolutionary novelties in known mineralized biological materials and might yield a better understanding of the processes of dentin formation and (hypo)mineralization in vertebrates, including humans.


Vulnerability to proactive semantic interference and progression to dementia among older adults with mild cognitive impairment.
There is evidence that vulnerability to proactive semantic interference may be an early manifestation of early Alzheimer's disease and other neurodegenerative disorders. At present, there is a paucity of data regarding the extent to which such deficits relate to the progression of cognitive deficits and to clinically significant endpoints such as dementia. In this study, we followed 76 older adults, initially diagnosed with mild cognitive impairment, for a period of up to 3 years. Twenty-seven of these individuals (35.5%) progressed from mild cognitive impairment to dementia. An examination of baseline neuropsychological performance indicated lower baseline scores for object memory among those progressing to dementia. However, baseline Mini-Mental State Examination scores, delayed memory for passages, delayed visual memory, letter fluency, category fluency, Trails B and Block Design did not differ among study groups. In contrast, the Semantic Interference Test, a measure susceptible to vulnerability to proactive semantic interference showed the greatest baseline differentiation between those who progressed and those who did not progress to dementia. Further, scores on this measure predicted future progression to dementia with high accuracy. Vulnerability to proactive interference may be an early manifestation of an early dementing process and may have utility in predicting future progression to dementia.